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Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process

Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process Ann Microbiol (2017) 67:405–416 DOI 10.1007/s13213-017-1272-4 ORIGINAL ARTICLE Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process 1 1 1 1,2 Hongyi Chen & Wenbiao Jin & Zhaoyun Liang & Abd El-Fatah Abomohra & 1 1 1 Xu Zhou & Renjie Tu & Songfang Han Received: 14 February 2017 /Accepted: 4 May 2017 /Published online: 26 May 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract Ammonia-oxidizing archaea (AOA) represent an im- Keywords Ammonia-oxidizing archaea . . portant group of ammonia-oxidizing microorganisms that are Ammonia-oxidizing bacteria Biological aerated filter able to convert ammonia to nitrite, a function which is crucial Environmental factors for the removal of nitrogen from wastewater. In this study, we investigated the abundance and diversity of AOA in a full-scale wastewater treatment plant (WWTP) which used a biological Introduction aerated filter (BAF) as the main processing mode. According to the quantitative PCR results, AOA clearly outnumbered Nitrification and denitrification are key steps in the removal of ammonia-oxidizing bacteria (AOB) during the whole process. biological nitrogen from wastewater (Ferrera and Sánchez 2016). The abundance of AOA amoA genes in the filter layer of BAF The oxidation of ammonia to nitrite is the first and rate-limiting 3 4 was highest with the value varied from 6.32 × 10 to 3.8 × 10 step in nitrification (Kowalchuk and Stephen 2001). Two groups copies/ng DNA. The highest abundance of AOB amoA genes of microorganisms, ammonia-oxidizing bacteria (AOB) and was 1.32 × 10 copies/ng DNA, recorded in the effluent of the ammonia-oxidizing archaea (AOA), are believed to be involved ACTIFLO® settling tank. The ratios of AOA/AOB in the in this process. Both AOB and AOA contain amoA genes that WWTP were maintained at two or three orders of magnitude. encode ammonia monooxygenase, the key enzyme that catalyzes Most AOA obtained from the WWTP fell within the the oxidation of ammonia to hydroxylamine (Li et al. 2016). Nitrosopumilus cluster. The abundance of AOA and AOB was AOA have been found in a broad variety of natural environ- significantly correlated with ammonium nitrogen concentrations ments, such as terrestrial systems (Chen et al. 2013), seawater and pH value. The community structure of AOA was signifi- (Santoro and Casciotti 2011;Houet al. 2013;Lv 2013), estuarine cantly influenced by dissolved oxygen concentrations, pH value and ocean sediments (Sakami 2012), suggesting the important and chemical oxygen demand. role of AOA in global nitrogen cycle. However, compared with AOB, little research has been conducted on AOA, especially on theroleofAOA in wastewater treatment processes. Environmental factors, such as ammonium nitrogen (NH -N) concentrations, temperature, salinity, pH and dis- solved oxygen (DO) levels can significantly affect the abun- * Wenbiao Jin jinwb@hit.edu.cn dance and diversity of AOA (Caffrey et al. 2007). Among these factors, DO level and NH -N concentration are consid- * Xu Zhou zhouxu@hit.edu.cn ered to be the most important (Yan et al. 2016). Ye and Zhang (2011) reported that DO level had a large effect on the types 1 and quantities of different AOA and AOB. Park et al. (2006) Shenzhen Engineering Laboratory of Microalgal Bioenergy, Harbin suggested that low DO levels might be the most appropriate Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China for the growth of AOA, but other studies have shown different results; for example, Kayee et al. (2011) identified a large Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt number of AOA amoA genes were found in wastewater 406 Ann Microbiol (2017) 67:405–416 treatment plants (WWTPs) at a DO concentration of 3.25 mg as the carbon source, which is necessary for denitrification. O /L. The substrate affinity (K ) valuesofAOA for NH -N The effluent from the BAF is finally treated by ACTIFLO® 2 s 4 are one to four logs lower than those values of AOB in settling tank for removal of the detached biofilm and SS. After WWTPs (Limpiyakorn et al. 2013), suggesting that AOA UV disinfection, the effluent is discharged into the river. The may be more suitable to grow under low NH -N concentra- sludge from the primary, ACTIFLO® and ACTIDYN® set- tions. These authors also reported that the expression of AOA tling tanks is introduced into the sludge treatment tank for amoA genes could be repressed under high NH -N treatment and compressed to sludge cake. The exhaust gas concentrations. generated during the treatment process is collected and The biological aerated filter (BAF) is a submerged waste- deodorized. water treatment technology which consists of porosity filters with a large surface area for the growth of biofilm (Bao et al. Sampling sites and physicochemical analysis 2016). Compared with conventional activated sludge process- es (Wang et al. 2017), the BAF has many advantages, includ- Five wastewater samples were collected from the WWTP, ing higher organic loadings, higher shock resistance and less including raw wastewater (RW), the influent of the BAF, the sludge production. In recent years the BAF has been widely effluent of BAF (EB), the effluent of the anaerobic filter (EA) used in WWTPs worldwide, especially for enhancing the re- and the effluent of the ACTIFLO® settling tank (EAS). Two moval of nitrogen from wastewater (Yu et al. 2016). filter samples (FB and FA) were taken from the filter layers of The presence of AOA in WWTPs has been reported by a the BAF and anaerobic filter tank, respectively. The total number of research groups (Park et al. 2006; Sonthiphand and height of the BAF was 7 m, and the filter layer, located in Limpiyakorn 2011;Tonget al. 2011; Yapsakli et al. 2011;Ye the middle of the BAF, was 3.5 m high. The inlet flow and and Zhang 2011; Gao et al. 2013;Chenet al. 2017), but the influent were located at the bottom of the BAF. In order to role AOA play in WWTP systems is still open to discussion. study the distribution of AOA communities in the BAF, we In this study, we selected a full-scale municipal WWTP with a collected seven samples from the filter layer in the BAF at a BAF as the main unit (Shenzhen, China) as the object of study. height of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 m, respectively. The abundance and diversity of AOA and AOB in different The concentrations ofCOD, SS, NH -N, nitrite nitrogen − − treatment stages of the WWTP were studied, with a focus on (NO -N), nitrate nitrogen (NO -N) and TN were analyzed 2 3 the AOA in the filter layer of the BAF. We also evaluated the according to standard methods (NEPA Chinese 2002). relationships between environmental factors and AOA Dissolved oxygen (DO) concentrations and pH values were communities. measured by using a DO meter (model YSI-550A; YSI Corp., Yellow Springs, OH) and pH meter (model FE20; Mettler- Toledo, Greifensee, Switzerland), respectively. Material and methods DNA extraction Description of the WWTP To achieve high concentrations of DNA, we filtered 1 L of Samples of wastewater and filters were taken from a full-scale each water sample through a cellulose nitrate membrane (pore 4 3 municipal WWTP with a treatment capacity of 5.0 × 10 m / size 0.22 μm). The filter was then cut into pieces, and these day in Shenzhen, China. The treatment processes of the pieces and glass beads were placed together into a centrifuge WWTP are a combination of BIOSTYR® BAF and tube and 10 mL of sterile water added. The mixture was gently ACTIFLO® setting tank (Veolia Water Technologies, Veolia mixed by vortex to ensure that all residues were separated Environnement, Paris, France). As shown in Fig. 1, the waste- from the membrane and then centrifuged at 4874 g for water is treated successively by passage through a bar screen, 5 min. For the filter samples, 10 mL of the filter suspension aerated grit chamber and primary settling tank to remove was put into a centrifuge tube together with 10 mL of sterile suspended solids (SS). The effluent from the primary settling water. The mixture was then gently mixed by vortex to ensure tank, the backwash wastewater following treatment in the that all biofilms had fallen offthe filters, followed by centrifu- ACTIDYN® settling tank (Veolia Water Technologies) and gation at 4874 g for 5 min. DNA was extracted from the the effluent from the anaerobic filter are introduced into the centrifuged deposit (0.5 g) by the Fast DNA® SPIN Kit For BAF for the removal of pollutants, including chemical oxygen Soil (MP Biomedicals, Santa Ana, CA) following the manu- demand (COD), NH -N, total nitrogen (TN) and total phos- facturer’s instructions. After being confirmed by conventional phorus (TP). Lime is added to the BAF to adjust the alkalinity. electrophoresis on a 1% agarose gel electrophoresis, the con- Of the effluent introduced into the BAF, 48% passes into an centration of DNA was measured on a NanoDrop UV-Vis anaerobic filter, which is used as denitrification tank for nitro- spectrophotometer (model ND-2000; Thermo Fisher gen removal. Methyl alcohol is added into the anaerobic filter Scientific, Waltham, MA). Ann Microbiol (2017) 67:405–416 407 Fig. 1 The flow chart of the targeted full-scale municipal wastewater treatment plant (WWTP) showing the main wastewater treatment processes and sampling sites. Water sampling sites (①– ⑤ and filter sampling sites (⑥,⑦) are shown. ① Raw wastewater,② influent of the biological aerated filter (BAF), ③ the effluent of BAF, ④ the effluent of the anaerobic filter tank, ⑤ the effluent of the ACTIFLO® settling tank, ⑥ filter layers of the BAF, ⑦ filter layers of the anaerobic filter tank. PAM Polyacrylamide, PAC polyaluminium chloride PCR amplification, sequencing and construction of clone primer pair Arch-amoA 26F/Arch-amoA 417R was used for libraries the AOA amoA genes, and the primer pair amoA-1F (5′- GGGGTTTCTACTGGTGGT-3′)/amoA-2R (5′-CCCC Primers A rch- amoA 26F (5 ′ -G AC T TCKGSAAAGCCTTCTTC-3′) (Rotthauwe et al. 1997) ACATMTTCTAYACWGAYTGGGC-3′)and Arch-amoA was selected for the AOB amoA genes. The quantification 417R (5′-GGKGTCATRTATGGWGGYAAYGTTGG-3′) reactions were performed in a qPCR system (LightCycler (Park et al. 2008) were selected to amplify the segment 1.5; Roche Applied Science, Penzburg, Germany). The re- of AOA amoA gene fragments. The PCR cycling program action mixture (20 μL) for each qPCR amplification consisted of an initial denaturation at 98 °C for 2 min, consisted of 10 μL SYBR Premix ExTaq™ II, 0.4 μLof followed by 35 cycles at 98 °C for 30 s, annealing at each primer, and 2 μL of template DNA. The purified PCR 55 °C for 30 s and 72 °C for 1 min, with a final extension products were ligated into pMD®19-T Vector and trans- at 72 °C for 5 min. Following purification with the formed into E. coli DH5α competent cells. The specificity MiniBest Agarose Gel DNA Extraction kit Ver.3.0 of each PCR product for the target genes was checked using (TaKaRa, Tokyo, Japan), the PCR products were ligated gel electrophoresis, and positive colonies were sent to The into the pMD 19-T vector and transformed into Beijing Genomics Institute for sequencing. After the target Escherichia coli DH5α competent cells following the man- genes had been confirmed, the plasmids DNA were extract- ufacturer’s instructions. ed from the positive colonies with the E.Z.N.A.™ plasmid The obtained sequences sharing at least 97% identity Mini Kit (Omega Bio-Tek Inc., Norcross, GA) and used as a were grouped into one operational taxonomic unit (OTU) standard sample. The mass concentration of the plasmid using Mothur. OTU-based parameters, including library DNA was determined on a NanoDrop UV-Vis spectropho- coverage (C), the Shannon–Weiner diversity index (H), tometer and converted into molecular units (the copy num- Pielou’sevennessindex (J), andthe Chao 1richnessesti- ber of the plasmids in a unit sample). The standard sample mate (S), were calculated following the methods described was diluted for eight different ten-fold dilutions and the in the previous studies (Good 1953;Chao 1987;Hillet al. threshold cycle (C ) value was determined; the standard 2003). The representative sequences were aligned to the curve between the C value and the logarithm of the sample database of the NCBI using the BLAST tool. Neighbor- concentration was then established. The qPCR conditions joining phylogenetic trees were constructed with the ob- were set at 95 °C for 30 s, followed by 40 cycles of tained similar sequences using MEGA 5.1 (bootstrap value 95 °C for 5 s, 55 °C for 30 s and 72 °C for 1 min; the was set at 1000 replicates). fluorescence value was read during each cycle at 72 °C. The dissolution curve was measured immediately after the PCR Quantification of AOA and AOB amoA genes reaction at 65–95 °C. The results were accurate when the melting temperature value was >80 °C, and the dissolution Quantitative PCR was employed to measure the abundance curve was a single peak. The correlation coefficients (R ) of AOA and AOB amoA gene copies in the samples. The were 0.9988 for AOA amoA and 0.9996 for AOB amoA. 408 Ann Microbiol (2017) 67:405–416 Statistical analysis treatment plants, with the abundance of AOA amoA genes being higher than that of AOB amoA genes in both the BAF The relationships between environmental factors and AOA and ACTIFLO® settling tank. AOA amoA genes were not abundance were evaluated by Pearson correlation coefficients detectable in the EA sample, possibly due to the low DO using SPSS version 17.0 (IBM Corp., Armonk, NY). The concentration in the anaerobic filter. The abundance of AOA links between AOA communities in the BAF and environ- amoA genes was highest in the FB sample (1.53 × 10 mental factors were assessed by redundancy analysis (RDA) copies/ng DNA). Among the wastewater samples, the EB using CANOCO for Windows 4.5 (Plant Research sample contained the highest amount of AOA amoA genes International, Wageningen, The Netherlands). (1.02 × 10 copies/ng DNA). The abundance of AOB amoA genes ranged between 0.54 copies/ng DNA and 1.32 × 10 copies/ng DNA, with the highest amount of AOB amoA genes Results found in the EAS sample. However, the abundance of AOA amoA genes in the two filter samples (FB and FA) was dis- Characteristics of the samples tinctly higher than that of AOB amoA genes. The AOA/AOB ratio in the FB and FA samples was 213.63 and 27.45, The water quality indexes of the collected samples are shown respectively. in Table 1. The concentrations of COD, NH -N and SS Since the abundance of AOA amoA genes in the FB sample sharply decreased following treatment in the primary settling was the highest, we investigated further the distribution of tank and BAF. The temperature variations among the different AOA in the filter layer of the BAF. The characteristics of units in the treatment system were negligible, with the tem- wastewater samples collected at different heights (0.5-m inter- perature ranging between 27.6 and 28.3 °C. However, the DO vals) of the filter layer were measured. As shown in Fig. 3,the concentrations of the five samples fluctuated greatly, with the recorded temperatures at the different heights were in the highest and lowest DO concentration recorded as 4.57 mg O / range of 28.4 to 29.1 °C. The highest pH value was 7.48, L in the EB sample and 0.67 mg O /L in the EA sample, which was recorded at a height of 2.0 m. The pH value de- respectively. The pH value ranged between 6.57 and 7.51. creased to pH 6.66 at a height of 4.0 m, but there was no significant change in pH value from 4 to 5 m in height. Abundance of AOA and AOB in the collected samples However, the concentration of DO increased from 2.12 to 5.78 mg O /L along the direction of filtration. As shown in Figure 2 shows the qPCR results of AOA and AOB amoA Fig. 4a, the NH -N concentration also significantly decreased genes of the collected samples. In general, the qPCR results from 11.98 mg N/L at a height of 2.0 to 2.57 mg N/L at 5 m. In indicated that AOB amoA genes were ubiquitous in the comparison, the NO -N concentration increased at different Table 1 Water quality indexes of the five water samples collected from the wastewater treatment plant Water samples F value P-value Physiochemical properties RW IB EB EA EAS a b c c c COD (mg O /L) 171.25 ± 8.92 48.05 ± 3.54 18.07 ± 3.20 18.75 ± 1.34 18.90 ± 4.37 533.2 0.0000 a ab bc de ce TN (mgN/L) 21.01 ± 2.71 15.50 ± 3.23 13.24 ± 2.68 4.96 ± 0.96 9.06 ± 1.24 20.6 0.0001 − a a b a a NO -N (mg N/L) 0.71 ± 0.23 2.73 ± 0.70 8.96 ± 2.27 2.30 ± 0.67 3.56 ± 1.21 19.5 0.0001 − a a a b a NO -N (mg N/L) 0.30 ± 0.05 0.50 ± 0.26 0.14 ± 0.06 3.42 ± 0.96 0.72 ± 0.42 23.6 0.0000 + a b c c c NH -N (mg N/L) 16.21 ± 2.82 10.61 ± 2.43 3.29 ± 0.68 3.86 ± 0.92 3.13 ± 1.14 30.9 0.0000 a b c c c SS (mg/L) 90 ± 17.35 44 ± 13.53 10 ± 2.65 7 ± 3.00 4 ± 3.46 38.3 0.0000 a b c c c pH 6.99 ± 0.12 7.51 ± 0.05 6.57 ± 0.03 6.73 ± 0.01 6.60 ± 0.03 126.5 0.0000 ab a c a bc DO (mg/L) 1.42 ± 0.22 0.94 ± 0.36 4.57 ± 0.89 0.67 ± 0.03 3.37 ± 1.36 14.8 0.0003 a a a a a Temperature (°C) 28.2 ± 0.10 27.6 ± 0.17 28.0 ± 0.06 27.9 ± 0.10 27.8 ± 0.82 1.1 0.4203 Values in the same row followed by the same lowcase letter are not significantly different at P ≤ 0.05 according to one-way analysis of variance followed by the post hoc Tukey tes) RW, Raw wastewater; IB, influent of the biological aeration filter; EB, effluent of the biological aerated filter (BAF); EA, effluent of the anaerobicfilter; EAS, effluent of ACTIFLO® setting tank b − − + COD, Chemical oxygen demand; TN, total nitrogen; NO -N, nitrate nitrogen; NO -N, nitrite nitrogen; NH -N, ammonium nitrogen; SS, soluble 3 2 4 solids; DO, dissolved oxygen DO concentration (mg/L) Ann Microbiol (2017) 67:405–416 409 - + - TN NO-N NH -N NO -N 3 4 2 AOA a) AOB 0.5 4 12 0.4 3 0.3 0.2 0.1 0.0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Height (m) RW IB EB EA EAS FB FA Samples COD b) Fig. 2 Quantitative analysis of ammonia-oxidizing archaea (AOA)and ammonia-oxidizing bacteria (AOB) amoA genes (encoding ammonia monooxygenase) in the collected samples. RW Raw wastewater, IB influent of the BAF, EB effluent of the BAF, EA effluent of anaerobic filter, EAS effluent of ACTIFLO® settling tank, FB samples taken from the filter layers of the BAF, FA samples taken from the anaerobic filter heights, with minimum and maximum concentrations of 3.64 and 12.07 mg N/L at heights of 2.0 and 5.0 m, respectively. Based on the low variations in TN and NO -N concentrations 2.0 2.5 3.0 3.5 4.0 4.5 5.0 at different heights, we deduced that NH -N was converted to Height (m) NO -N and that the nitrification reactions mainly occurred in Fig. 4 Water quality indexes of the effluent of filter samples collected at the filter layers. The occurrence of nitrification reactions in the different heights of the BAF. a Concentration of total nitrogen (TN), filter layer could also provide a possible explanation for the + − ammonium nitrogen (NH -N), nitrite nitrogen (NO -N) and nitrate 4 2 decrease in pH value. The COD concentration decreased nitrogen (NO -N) b Concentration of chemical oxygen demand (COD) sharply from 161.02 to 46.85 mg/L with increasing height of the BAF filter layers to 5 m (Fig. 4b). at a height of 5.0 m of the BAF. In comparison, the amount of The abundances of AOA and AOB in the filter samples at AOB amoA genes found in the BAF varied between 20.64 and different heights of the BAF are shown in Fig. 5. The abun- 1.05 × 10 copies/ng DNA, with the maximum values record- dance of AOA amoA genes varied between 6.32 × 10 and ed at a height of 4.0 m of the BAF. The ratios of AOA/AOB 3.8 × 10 copies/ng DNA, while maximum value was attained ranged between 845.37 and 2784.91, which indicated that AOAwere dominant over AOB in the BAF. The water quality clearly improved with increasing height along the BAF. Temperture pH DO concentration Diversity of AOA communities The distribution and relative abundance of AOA amoA genes in the WWTP was investigated. Based on AOA amoA gene sequences, we constructed and evaluated five clone libraries and 27 OTUs. The distribution of the relative abundances of OTUs in the collected samples are shown in Fig 6.OTU4 and 22 OTU8 were dominant in the RW and EAS samples, suggest- ing that the AOA communities in the influent and effluent of the WWTP changed little. OTU3 was dominant in FB and FA, 20 1 2.0 2.5 3.0 3.5 4.0 4.5 5.0 which implied that the BAF and anaerobic filter shared the Height (m) same dominant OTU type. OTU3 was also the most widely Fig. 3 Changes in the temperature, pH and dissolved oxygen (DO) distributed OTU type, being present in all five samples tested. concentration of the effluent of filter samples collected at different heights of the BAF The results suggested that OTU3 had a strong adaptability to Temperature (ºC) lg(amoA genes copies) pH COD concentration (mg O /L) TN concentration (mg N/L) AOA/AOB 410 Ann Microbiol (2017) 67:405–416 AOA AOB AOA/AOB Fig. 5 The abundance of AOA 120 3000 and AOB amoA genes in filter 40,000 samples collected at different heights of the BAF 35,000 30,000 25,000 60 1500 20,000 15,000 10,000 5,000 0 0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Height (m) the environment. Although OTU4 dominated in the RW sam- all higher than the samples from the filers (FB and FA), indi- ple, it was not present in the FB sample, suggesting that the cating that the AOA from wastewater had a richer species environmental conditions in the BAF might be not suitable for diversity. The highest H and S values were 2.47 and 34.5, the growth of OTU4. The C, J, H and S values of the AOA respectively, detected from the EAS sample (Table 2). amoA gene clone libraries are presented in Table 2. The results The distribution and relative abundance of AOA amoA showed that RW and EAS contained 12 and 10 OTU types, genes in the filter layer of the BAF was further investigated respectively. In contrast, only five OTU types were detected in (Fig. 7). Six clone libraries were constructed and eight OTUs the FA sample. The FA sample had the minimum H value were obtained from the AOA amoA gene sequences at the 3% (0.54) among the five samples measured, suggesting that the nucleotide cut-off. The relative abundance of OTUs in the diversity of AOA amoA genes in the filter layer of the anaer- samples collected from the heights of 2.0 and 3.5 m was over obic filter was lower than in the other samples. The C value of 60%, and OTUb and OTUc were the dominant types in sam- the FB and FA samples was 90.0 and 90.6%, respectively, ples collected between the heights of 4.0 and 5.0 m. The which indicated that clone libraries covered the AOA commu- relative abundance of both kinds of OTU types was around nities in FA and FB samples and that the majority of species 40%. OTUb was the most prevalent OTU type among the could be detected and identified. For the samples taken from eight OTUs in the BAF, being present in all samples wastewater (RW, EB and EAS), the H value and S value were (Fig. 7). The value of C ranged between 78.6 and 100%, OTU27 100% Fig. 6 Distribution and relative OTU26 abundance of the AOA amoA OTU25 gene in the collected samples. 90% OTU24 OTU Operational taxonomic unit OYU23 OTU22 80% OTU21 OTU20 OTU19 70% OTU18 OTU17 60% OTU16 OTU15 OTU14 50% OTU13 OTU12 40% OTU11 OTU10 OTU9 30% OTU8 OTU7 OTU6 20% OTU5 OTU4 10% OTU3 OTU2 OTU1 0% RW EB FB FA EAS Samples AOB amoA genes (copies/ng DNA) Percentage AOA amoA genes (copies/ng DNA) Ann Microbiol (2017) 67:405–416 411 Table 2 Diversity of each clone library of ammonia oxidizing archaea Table 3 Diversity of each clone library of ammonia oxidizing archaea amoA genes in the five water samples amoA genes at different heights of the biological aerated filter Samples No. of clones OTUs CJ H S Height (m) No. of clones OTUsCJ H S RW 37 12 78.4% 0.65 1.63 21.78 2 9 2 100% 6.770.642.00 EB 18 8 68.2% 0.85 1.95 11.50 2.5 23 3 100% 0.69 0.75 3.00 FB 29 7 90.0% 0.67 1.30 8.88 3 14 4 78.6% 0.540.758.5 FA 32 5 90.6% 0.34 0.54 6.88 3.5 24 4 95.8% 0.69 0.96 4.50 EAS 22 10 77.8% 1.19 2.47 34.5 4 7 3 85.7% 0.91 1.00 3.50 5 28 6 92.8% 0.751.356.11 OTU, Operational taxonomic unit; C, library coverage; H, Shannon– Weiner diversity index; J,Pielou’s evenness index; S Chao 1 richness estimate phylogenetically within the Nitrosopumilus cluster (Jung et al. 2011). OTU8, another dominant OTU type in the RW suggesting that AOA communities covered the whole clone and EAS samples, correlated with sequences collected from libraries of these samples (Table 3). However, the H value the Mississippi River. OTU10 in the EAS sample was seen to increased gradually from 0.64to1.35withincreasingof be closely related to the sequences from WWTPs, which also height from 2.0 to 5.0 m, indicating that the increase of belong to the Nitrosopumilus cluster (Bai et al. 2012; Gao AOA diversity was related to the height of filter layer in BAF. et al. 2014). OTU3, which was dominant in the filter layer of BAF, showed a similarity to the AOA species detected in bench-scale airlift reactor for wastewater treatment. OTU26 Phylogenetic analysis of AOA was strongly correlated with AOA sequences collected from The phylogenetic tree of AOA amoA genes in the municipal rice soil. OTU14 from the FB sample showed a high similarity to sequence WG6612 from Yellowstone hot spring (Zhang WWTPs is shown in Fig. 8. Based on the results, the obtained AOA amoA sequences could be grouped into two major clus- et al. 2008). OTU1 was present in the EB, FB and FA samples and was correlated to SL-37 from the Stanley WWTP that ters. Most of the OTUs in one of the major clusters were strongly correlated with sequences from the natural environ- falls into the Nitrososphaera sister subcluster (Zhang et al. 2009). ment, such as rivers, lakes, estuary sediments and soils. The OTUs in the second major cluster had many similarities to the Eight OTUs also fell into two major clusters, as shown in the AOA phylogenetic tree of the BAF (Fig. 9). OTUd, which sequences from WWTPs. The most abundant OTU type in the RW and EAS samples, OTU4, showed high similarities to the was the dominant OTU type in the BAF filter between the sequence HZNAOA7 from Dongjiang River sediment, which height at 2.0 and 3.5 m, was strongly correlated with the se- quences belonging to sediments of Dongjiang River. OTUb belongs to Nitrosopumilus cluster (Sun et al. 2013). OTU27 from the EB sample was related to the sequences of and OTUc, the dominant types in the BAF between 4.0 and 5.0 m, were similar to the AOA clones detected in a Nitrosopumilaceae archaeon MY1 which fall 100% Fig. 7 Distribution and relative abundance of AOA amoA gene of 90% the filter sample at different OTUh heights of the BAF 80% OTUg OTUf 70% OTUe 60% OTUd 50% OTUc 40% OTUb OTUa 30% 20% 10% 0% 2 2.5 3 3.5 4 5 Height (m) Precentage 412 Ann Microbiol (2017) 67:405–416 Fig. 8 Phylogenetic tree of AOA amoA gene sequence from different samples in the municipal wastewater treatment plants Ann Microbiol (2017) 67:405–416 413 Fig. 9 Phylogenetic tree of AOA amoA gene sequences from different samples in the filter layer of the BAF wastewater treatment bioreactor operating at 30 °C for Discussion 10 days. Most OTUs obtained from the filter layer of BAF fell into the Nitrosocomicus cluster. These results led us to A number of studies have demonstrated the presence of AOA conclude that the AOA near the inlet of the BAF were similar amoA genes in WWTPs, but the abundances of AOA and to the ones from natural environments, while the AOA near AOB amoA genes have remained a focus of controversy. the outlet of BAF were similar to the ones from wastewater AOB amoA genes have been reported to be the dominant type treatment systems. in WWTPs (Wells et al. 2009; Jin et al. 2010;Tongetal. 2011; Bai et al. 2012; Gao et al. 2013). Yapsakli et al. (2011)inves- tigated AOA and AOB abundance in a full-scale leachate Correlations between AOA community structure and environmental factors We used RDA to evaluate the correlations between the AOA community structures in the BAF and environmental factors. The results are shown in Fig. 10, where Axis1 and Axis2 correspond to 73.0 and 10.9% of the cumulative variance of the AOA community–environment relationship, respectively. The location of the red arrows of COD, pH and DO near Axis1 suggest that these factors had significant effects on the community structures of AOA from the filter layer of the BAF. The AOA near the bottom of filter layer (between the height of 2 and 2.5 m) in the BAF were mainly distributed along pH and DO gradients, indicating that the AOA from the bottom of the filter layer were strongly correlated with pH and DO concentrations. The communities of AOA at the top of the filter layer (at the height of 5 m) in the BAF were significantly + − affected by COD, NH -N and NO -N. In addition, the DO 4 2 concentration showed a strong negative correlation with the + − + Fig. 10 Ordination plots generated by redundancy analysis. Red arrows concentrations of COD, NH -N and NO -N. NH -N con- 4 2 4 − − Environmental variables (pH, DO, COD, TN, NO -N, NO -N, and 2 3 centration also showed a strong negative correlation with NH -N), circles and numbers filter samples collected at different heights from the BAF NO -N concentration. 3 414 Ann Microbiol (2017) 67:405–416 treatment plant with high concentration of COD (5000 mg O / wastewater. It has been suggested that some types of AOA L) and TN (2000 mg N/L) and found that AOB were dominant may only be able to survive under low DO concentrations; in the treatment plant, with an AOB/AOA ratio range of one to thus low DO concentrations may be a crucial factor for the three orders of magnitude. These results were confirmed by presence of AOA (Erguder et al. 2009). However, the results Gao et al. (2014) who reported that AOB amoA genes obtained in our study are no in agreement with this specula- outnumbered AOA in ten WWTPs. In contrast, Kayee et al. tion. In our study, the amount of AOA amoA genes increased (2011)found that AOA amoA genes outnumbered AOB amoA with increasing DO concentration in the filter layer. One pos- genes in eight municipal WWTPs, and Bai et al. (2012)dem- sible explanation for this phenomenon is the possibility that onstrated that AOA were dominant in three municipal the AOA types in the BAF were able to adapt to the environ- WWTPs, while AOB were dominant in three industrial ment of a relatively high DO level. It is also possible that the WWTPs. A relatively higher amount of AOA amoA genes oxygen was consumed quickly by heterotrophic microorgan- was also found by Muβmann et al. (2011)aswellas isms on the surface of biofilm and that AOA may be present in Sonthiphand and Limpiyakorn (2011). In the present study, the depths of biofilm at low DO levels. In addition, with the we investigated a full-scale municipal WWTP with a BAF increase in the height of the BAF from 2.0 to 5.0 m, COD and as the main processing unit. The WWTP had a high capacity NH -N concentrations decreased while at the same time there for treating pollutants, with the concentrations of COD and SS was a significant increase of AOA amoA genes. This phenom- in wastewater decreasing from 171.25 mg O /L and 90 mg/L enon suggests that low COD and NH -N levels may be fa- 2 4 to 18.90 mg O /L and 4 mg/L, respectively (Table 1). AOA vorable for the growth of AOA in BAF. DO concentrations and AOB amoA genes were detected in the collected samples and pH values, which changed regularly along the direction of from different sections of the WWTP, and our results showed filtration, may also be important influencing factors. that AOA were dominant in the BAF processing unit. Across AOA have been divided into five major clusters based on the flow path of the treatment system, the abundance of AOA the published AOA amoA gene sequences (Pester et al. 2012): amoA genes in wastewater increased from 26.00 to 217.27 Nitosopumilis cluster, Nitrososphaera cluster, Nitrosocaldus copies/ng DNA, and the NH -N concentration decreased cluster, Nitrosotalea cluster and Nitrosophaera sister cluster. from 16.21 to 3.13 mg N/L (Table 1). The abundance of In the present study, five clone libraries of AOA were con- AOB amoA genes in the wastewater also increased from structed with samples from the WWTP. The obtained AOA 4.18 to 133.97 copies/ng DNA during the process. amoA gene sequences fell into two major clusters. OTU4 and AOA abundance showed a strong, positive association OTU8, which were dominant in the wastewater samples, with temperature and NO -N concentration and a significant, grouped into Cluster A, showing a highly similarity with se- + − negative correlation with NH -N and NO -N concentra- quences from natural environments, which belonged to the 4 2 tions, respectively, and pH value (Table 4). Since NO -N Nitrosopumilus cluster. OTU3 from FB fell into Cluster B, was mainly converted from NH -N oxidation in the BAF, with most sequences obtained from WWTPs. Many studies NO -N concentrations increased concomitantly with the de- have found that most AOA obtained from WWTPs belong to + − crease in NH -N concentrations. In addition, NO -N con- the Nitrososphaera cluster (Muβmann et al. 2011;Tongetal. 4 2 centrations were always very low, at <0.4 mg N/L. The pH 2011; Yapsakli et al. 2011; Sauder et al. 2012; Gao et al. value and NH -N concentration decreased with increasing 2013), but other studies have reported that Nitrosopumilus height of the filter, with a simultaneous increase in AOA and cluster is dominant in WWTPs. A novel AOA strain SAT1 AOB amoA genes. We therefore surmised that NH -N con- enriched from activated sludge has been affiliated with the centration and pH value were the main critical influences on Nitrosopumilus cluster (Li et al. 2016). Similar results were the abundance of AOA. In terms of AOB abundance, the pH also reported by Wu et al. (2013). value and NH -N concentration had a significant negative The abundance and diversity of AOA amoA genes can be influence. As such, AOA and AOB amoA genes shared com- significantly influenced by environmental factors. The in- mon influencing factors. As an electron acceptor, DO is also crease in DO concentrations could promote the degradation considered to be an important factor in the processing of of organic pollutants, and the nitrification process in which Table 4 Statistical analysis of physicochemical parameters and the abundance of amoA genes of AOA and AOB + − − Parameters NH -N NO -N NO -N TN COD DO pH Temperature 4 2 3 AOA amoA genes −0.685** −0.623** 0.536** −0.116 −0.252 0.355 −0.555** 0.596** AOB amoA genes −0.450* −0.316 0.166 −0.387 −0.078 0.380* −0.572** 0.226 **Highly significant at P ≤ 0.01; *significant at P ≤ 0.05 Ann Microbiol (2017) 67:405–416 415 + − − high carbon to nitrogen ratios under micro-aerobic condition. NH -N and NO -N is converted to NO -N correlated neg- 4 2 3 Biores Technol 232:417–422 + − atively to the concentrations of COD, NH -N and NO -N. 4 2 Erguder TH, Boon N, Wittebolle L, Marzorati M, Verstraete W (2009) + − The negative correlation between NH -N and NO -N may 4 3 Environmental factors shaping the ecological niches of ammonia- + − be attributed to the conversion of NH -N into NO -N in the oxidizing archaea. FEMS Microbiol Rev 33(5):855–869 4 3 Ferrera I, Sánchez O (2016) Insights into microbial diversity in wastewa- BAF. The minimal angles between DO, pH, COD and Axis 1 ter treatment systems: how far have we come? Biotechnol Adv 34: also confirmed that the AOA community structure in BAF 790–802 was significantly influenced by DO concentrations, pH value Gao J, Luo X, Wu G, Li T, Peng Y (2014) Abundance and diversity based and COD concentrations (Fig. 10). on amoA genes of ammonia-oxidizing archaea and bacteria in ten wastewater treatment systems. Appl Microbiol Biotechnol 98:3339– In summary, in this study, we investigated the abundance and diversity of AOA in a full-scale WWTP with BAF as the Gao JF, Luo X, Wu GX, Li T, Peng YZ (2013) Quantitative analyses of main processing unit. We found that AOA amoA genes the composition and abundance of ammonia-oxidizing archaea and outnumbered AOB amoA genes in the WWTP, especially in ammonia-oxidizing bacteria in eight full-scale biological wastewater the filter layer of the BAF. The abundance of AOA amoA treatment plants. Bioresour Technol 138C:285–296 3 4 Good IJ (1953) The population frequencies of species and the estimation genes in the BAF varied from 6.32 × 10 to 3.8 × 10 of population parameters. Biometrika 40:237–264 copies/ng DNA, while the highest abundance of AOB amoA Hill TC, Walsh KA, Harris JA, Moffett BF (2003) Using ecological genes was 1.05 × 10 copies/ng DNA. AOAwere dominant in diversity measures with bacterial communities. FEMS Microbiol the BAF, suggesting the important role of this type of micro- Ecol 43:1–11 Hou J, Song C, Cao X, Zhou Y (2013) Shifts between ammonia- organisms on nitrogen removal. The AOA sequences obtained oxidizing bacteria and archaea in relation to nitrification potential from the WWTP were grouped into two major clusters, with across trophic gradients in two large Chinese lakes (Lake Taihu and one cluster strongly correlating with AOA from natural envi- Lake Chaohu). Water Res 47:2285–2296 ronments and the other cluster showing a high similarity to the Jin T, Zhang T, Yan Q (2010) Characterization and quantification of AOA from WWTPs. The AOA in the WWTP were mostly ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR. Appl related to the Nitrosopumilus cluster. pH and NH -N were Microbiol Biotechnol 87:1167–1176 critical environmental factors with a major effect on the abun- Jung MY, Park SJ, Min D, Kim JS, Rijpstra WIC, Damsté JSS, Kim GJ, dance of AOA and AOB amoA genes. AOA community struc- Madsen EL, Rhee SK (2011) Enrichment and characterization of an ture was significantly influenced by DO concentrations, pH autotrophic ammonia-oxidizing archaeon of mesophilic Crenarchaeal group I.1a from an agricultural soil. Appl Environ value and COD concentration. Microbiol 77:8635–8647 Kayee P, Sonthiphand P, Rongsayamanont C, Limpiyakorn T (2011) Acknowledgments This work was supported by the National Natural Archaeal amoA genes outnumber bacterial amoA genes in munici- Science Foundation of China (No.50978069). pal wastewater treatment plants in Bangkok. Microb Ecol 62:776– 788 (retracted article) Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: a mod- Compliance with ethical standards el for molecular microbial ecology. Annu Rev Microbiol 55:485– Disclosure No conflict of interest was reported by the authors. Li Y, Ding K, Wen X, Zhang B, Shen B, Yang Y (2016) A novel ammonia-oxidizing archaeon from wastewater treatment plant: its enrichment, physiological and genomic characteristics. Sci Rep Uk 6:23747 References Limpiyakorn T, Fürhacker M, Haberl R, Chodanon T, Srithep P, Sonthiphand P (2013) amoA-encoding archaea in wastewater treat- Bai Y, Sun Q, Wen D, Tang X (2012) Abundance of ammonia-oxidizing ment plants: a review. Appl Microbiol Biotechnol 97:1425–1439 bacteria and archaea in industrial and domestic wastewater treatment Lv ZLWJ (2013) Abundance and diversity of ammonia-oxidizing archaea systems. 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Extremophiles 12: Enhanced performance of denitrifying sulfide removal process at 605–615 416 Ann Microbiol (2017) 67:405–416 Pester M, Rattei T, Flechl S, Gröngröft A, Richter A, Overmann J, Wang Q, Wei W, Gong Y, Yu Q, Li Q, Sun J, Yuan Z (2017) Technologies for reducing sludge production in wastewater treatment plants: State Reinhold-Hurek B, Loy A, Wagner M (2012) amoA-based consen- sus phylogeny of ammonia-oxidizing archaea and deep sequencing of the art. Sci Total Environ of amoA genes from soils of four different geographic regions. Wells GF, Park HD, Yeung CH, Eggleston B, Francis CA, Criddle CS Environ Microbiol 14:525–539 (2009) Ammonia-oxidizing communities in a highly aerated full- scale activated sludge bioreactor: betaproteobacterial dynamics and Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia low relative abundance of Crenarchaea. 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Appl Microbiol Biotechnol 91:1215–1225 a laboratory scale reactor and two wastewater treatment plants. J Appl Microbiol 107:970–977 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process

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Springer Journals
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Copyright © 2017 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-017-1272-4
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Abstract

Ann Microbiol (2017) 67:405–416 DOI 10.1007/s13213-017-1272-4 ORIGINAL ARTICLE Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process 1 1 1 1,2 Hongyi Chen & Wenbiao Jin & Zhaoyun Liang & Abd El-Fatah Abomohra & 1 1 1 Xu Zhou & Renjie Tu & Songfang Han Received: 14 February 2017 /Accepted: 4 May 2017 /Published online: 26 May 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract Ammonia-oxidizing archaea (AOA) represent an im- Keywords Ammonia-oxidizing archaea . . portant group of ammonia-oxidizing microorganisms that are Ammonia-oxidizing bacteria Biological aerated filter able to convert ammonia to nitrite, a function which is crucial Environmental factors for the removal of nitrogen from wastewater. In this study, we investigated the abundance and diversity of AOA in a full-scale wastewater treatment plant (WWTP) which used a biological Introduction aerated filter (BAF) as the main processing mode. According to the quantitative PCR results, AOA clearly outnumbered Nitrification and denitrification are key steps in the removal of ammonia-oxidizing bacteria (AOB) during the whole process. biological nitrogen from wastewater (Ferrera and Sánchez 2016). The abundance of AOA amoA genes in the filter layer of BAF The oxidation of ammonia to nitrite is the first and rate-limiting 3 4 was highest with the value varied from 6.32 × 10 to 3.8 × 10 step in nitrification (Kowalchuk and Stephen 2001). Two groups copies/ng DNA. The highest abundance of AOB amoA genes of microorganisms, ammonia-oxidizing bacteria (AOB) and was 1.32 × 10 copies/ng DNA, recorded in the effluent of the ammonia-oxidizing archaea (AOA), are believed to be involved ACTIFLO® settling tank. The ratios of AOA/AOB in the in this process. Both AOB and AOA contain amoA genes that WWTP were maintained at two or three orders of magnitude. encode ammonia monooxygenase, the key enzyme that catalyzes Most AOA obtained from the WWTP fell within the the oxidation of ammonia to hydroxylamine (Li et al. 2016). Nitrosopumilus cluster. The abundance of AOA and AOB was AOA have been found in a broad variety of natural environ- significantly correlated with ammonium nitrogen concentrations ments, such as terrestrial systems (Chen et al. 2013), seawater and pH value. The community structure of AOA was signifi- (Santoro and Casciotti 2011;Houet al. 2013;Lv 2013), estuarine cantly influenced by dissolved oxygen concentrations, pH value and ocean sediments (Sakami 2012), suggesting the important and chemical oxygen demand. role of AOA in global nitrogen cycle. However, compared with AOB, little research has been conducted on AOA, especially on theroleofAOA in wastewater treatment processes. Environmental factors, such as ammonium nitrogen (NH -N) concentrations, temperature, salinity, pH and dis- solved oxygen (DO) levels can significantly affect the abun- * Wenbiao Jin jinwb@hit.edu.cn dance and diversity of AOA (Caffrey et al. 2007). Among these factors, DO level and NH -N concentration are consid- * Xu Zhou zhouxu@hit.edu.cn ered to be the most important (Yan et al. 2016). Ye and Zhang (2011) reported that DO level had a large effect on the types 1 and quantities of different AOA and AOB. Park et al. (2006) Shenzhen Engineering Laboratory of Microalgal Bioenergy, Harbin suggested that low DO levels might be the most appropriate Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China for the growth of AOA, but other studies have shown different results; for example, Kayee et al. (2011) identified a large Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt number of AOA amoA genes were found in wastewater 406 Ann Microbiol (2017) 67:405–416 treatment plants (WWTPs) at a DO concentration of 3.25 mg as the carbon source, which is necessary for denitrification. O /L. The substrate affinity (K ) valuesofAOA for NH -N The effluent from the BAF is finally treated by ACTIFLO® 2 s 4 are one to four logs lower than those values of AOB in settling tank for removal of the detached biofilm and SS. After WWTPs (Limpiyakorn et al. 2013), suggesting that AOA UV disinfection, the effluent is discharged into the river. The may be more suitable to grow under low NH -N concentra- sludge from the primary, ACTIFLO® and ACTIDYN® set- tions. These authors also reported that the expression of AOA tling tanks is introduced into the sludge treatment tank for amoA genes could be repressed under high NH -N treatment and compressed to sludge cake. The exhaust gas concentrations. generated during the treatment process is collected and The biological aerated filter (BAF) is a submerged waste- deodorized. water treatment technology which consists of porosity filters with a large surface area for the growth of biofilm (Bao et al. Sampling sites and physicochemical analysis 2016). Compared with conventional activated sludge process- es (Wang et al. 2017), the BAF has many advantages, includ- Five wastewater samples were collected from the WWTP, ing higher organic loadings, higher shock resistance and less including raw wastewater (RW), the influent of the BAF, the sludge production. In recent years the BAF has been widely effluent of BAF (EB), the effluent of the anaerobic filter (EA) used in WWTPs worldwide, especially for enhancing the re- and the effluent of the ACTIFLO® settling tank (EAS). Two moval of nitrogen from wastewater (Yu et al. 2016). filter samples (FB and FA) were taken from the filter layers of The presence of AOA in WWTPs has been reported by a the BAF and anaerobic filter tank, respectively. The total number of research groups (Park et al. 2006; Sonthiphand and height of the BAF was 7 m, and the filter layer, located in Limpiyakorn 2011;Tonget al. 2011; Yapsakli et al. 2011;Ye the middle of the BAF, was 3.5 m high. The inlet flow and and Zhang 2011; Gao et al. 2013;Chenet al. 2017), but the influent were located at the bottom of the BAF. In order to role AOA play in WWTP systems is still open to discussion. study the distribution of AOA communities in the BAF, we In this study, we selected a full-scale municipal WWTP with a collected seven samples from the filter layer in the BAF at a BAF as the main unit (Shenzhen, China) as the object of study. height of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 m, respectively. The abundance and diversity of AOA and AOB in different The concentrations ofCOD, SS, NH -N, nitrite nitrogen − − treatment stages of the WWTP were studied, with a focus on (NO -N), nitrate nitrogen (NO -N) and TN were analyzed 2 3 the AOA in the filter layer of the BAF. We also evaluated the according to standard methods (NEPA Chinese 2002). relationships between environmental factors and AOA Dissolved oxygen (DO) concentrations and pH values were communities. measured by using a DO meter (model YSI-550A; YSI Corp., Yellow Springs, OH) and pH meter (model FE20; Mettler- Toledo, Greifensee, Switzerland), respectively. Material and methods DNA extraction Description of the WWTP To achieve high concentrations of DNA, we filtered 1 L of Samples of wastewater and filters were taken from a full-scale each water sample through a cellulose nitrate membrane (pore 4 3 municipal WWTP with a treatment capacity of 5.0 × 10 m / size 0.22 μm). The filter was then cut into pieces, and these day in Shenzhen, China. The treatment processes of the pieces and glass beads were placed together into a centrifuge WWTP are a combination of BIOSTYR® BAF and tube and 10 mL of sterile water added. The mixture was gently ACTIFLO® setting tank (Veolia Water Technologies, Veolia mixed by vortex to ensure that all residues were separated Environnement, Paris, France). As shown in Fig. 1, the waste- from the membrane and then centrifuged at 4874 g for water is treated successively by passage through a bar screen, 5 min. For the filter samples, 10 mL of the filter suspension aerated grit chamber and primary settling tank to remove was put into a centrifuge tube together with 10 mL of sterile suspended solids (SS). The effluent from the primary settling water. The mixture was then gently mixed by vortex to ensure tank, the backwash wastewater following treatment in the that all biofilms had fallen offthe filters, followed by centrifu- ACTIDYN® settling tank (Veolia Water Technologies) and gation at 4874 g for 5 min. DNA was extracted from the the effluent from the anaerobic filter are introduced into the centrifuged deposit (0.5 g) by the Fast DNA® SPIN Kit For BAF for the removal of pollutants, including chemical oxygen Soil (MP Biomedicals, Santa Ana, CA) following the manu- demand (COD), NH -N, total nitrogen (TN) and total phos- facturer’s instructions. After being confirmed by conventional phorus (TP). Lime is added to the BAF to adjust the alkalinity. electrophoresis on a 1% agarose gel electrophoresis, the con- Of the effluent introduced into the BAF, 48% passes into an centration of DNA was measured on a NanoDrop UV-Vis anaerobic filter, which is used as denitrification tank for nitro- spectrophotometer (model ND-2000; Thermo Fisher gen removal. Methyl alcohol is added into the anaerobic filter Scientific, Waltham, MA). Ann Microbiol (2017) 67:405–416 407 Fig. 1 The flow chart of the targeted full-scale municipal wastewater treatment plant (WWTP) showing the main wastewater treatment processes and sampling sites. Water sampling sites (①– ⑤ and filter sampling sites (⑥,⑦) are shown. ① Raw wastewater,② influent of the biological aerated filter (BAF), ③ the effluent of BAF, ④ the effluent of the anaerobic filter tank, ⑤ the effluent of the ACTIFLO® settling tank, ⑥ filter layers of the BAF, ⑦ filter layers of the anaerobic filter tank. PAM Polyacrylamide, PAC polyaluminium chloride PCR amplification, sequencing and construction of clone primer pair Arch-amoA 26F/Arch-amoA 417R was used for libraries the AOA amoA genes, and the primer pair amoA-1F (5′- GGGGTTTCTACTGGTGGT-3′)/amoA-2R (5′-CCCC Primers A rch- amoA 26F (5 ′ -G AC T TCKGSAAAGCCTTCTTC-3′) (Rotthauwe et al. 1997) ACATMTTCTAYACWGAYTGGGC-3′)and Arch-amoA was selected for the AOB amoA genes. The quantification 417R (5′-GGKGTCATRTATGGWGGYAAYGTTGG-3′) reactions were performed in a qPCR system (LightCycler (Park et al. 2008) were selected to amplify the segment 1.5; Roche Applied Science, Penzburg, Germany). The re- of AOA amoA gene fragments. The PCR cycling program action mixture (20 μL) for each qPCR amplification consisted of an initial denaturation at 98 °C for 2 min, consisted of 10 μL SYBR Premix ExTaq™ II, 0.4 μLof followed by 35 cycles at 98 °C for 30 s, annealing at each primer, and 2 μL of template DNA. The purified PCR 55 °C for 30 s and 72 °C for 1 min, with a final extension products were ligated into pMD®19-T Vector and trans- at 72 °C for 5 min. Following purification with the formed into E. coli DH5α competent cells. The specificity MiniBest Agarose Gel DNA Extraction kit Ver.3.0 of each PCR product for the target genes was checked using (TaKaRa, Tokyo, Japan), the PCR products were ligated gel electrophoresis, and positive colonies were sent to The into the pMD 19-T vector and transformed into Beijing Genomics Institute for sequencing. After the target Escherichia coli DH5α competent cells following the man- genes had been confirmed, the plasmids DNA were extract- ufacturer’s instructions. ed from the positive colonies with the E.Z.N.A.™ plasmid The obtained sequences sharing at least 97% identity Mini Kit (Omega Bio-Tek Inc., Norcross, GA) and used as a were grouped into one operational taxonomic unit (OTU) standard sample. The mass concentration of the plasmid using Mothur. OTU-based parameters, including library DNA was determined on a NanoDrop UV-Vis spectropho- coverage (C), the Shannon–Weiner diversity index (H), tometer and converted into molecular units (the copy num- Pielou’sevennessindex (J), andthe Chao 1richnessesti- ber of the plasmids in a unit sample). The standard sample mate (S), were calculated following the methods described was diluted for eight different ten-fold dilutions and the in the previous studies (Good 1953;Chao 1987;Hillet al. threshold cycle (C ) value was determined; the standard 2003). The representative sequences were aligned to the curve between the C value and the logarithm of the sample database of the NCBI using the BLAST tool. Neighbor- concentration was then established. The qPCR conditions joining phylogenetic trees were constructed with the ob- were set at 95 °C for 30 s, followed by 40 cycles of tained similar sequences using MEGA 5.1 (bootstrap value 95 °C for 5 s, 55 °C for 30 s and 72 °C for 1 min; the was set at 1000 replicates). fluorescence value was read during each cycle at 72 °C. The dissolution curve was measured immediately after the PCR Quantification of AOA and AOB amoA genes reaction at 65–95 °C. The results were accurate when the melting temperature value was >80 °C, and the dissolution Quantitative PCR was employed to measure the abundance curve was a single peak. The correlation coefficients (R ) of AOA and AOB amoA gene copies in the samples. The were 0.9988 for AOA amoA and 0.9996 for AOB amoA. 408 Ann Microbiol (2017) 67:405–416 Statistical analysis treatment plants, with the abundance of AOA amoA genes being higher than that of AOB amoA genes in both the BAF The relationships between environmental factors and AOA and ACTIFLO® settling tank. AOA amoA genes were not abundance were evaluated by Pearson correlation coefficients detectable in the EA sample, possibly due to the low DO using SPSS version 17.0 (IBM Corp., Armonk, NY). The concentration in the anaerobic filter. The abundance of AOA links between AOA communities in the BAF and environ- amoA genes was highest in the FB sample (1.53 × 10 mental factors were assessed by redundancy analysis (RDA) copies/ng DNA). Among the wastewater samples, the EB using CANOCO for Windows 4.5 (Plant Research sample contained the highest amount of AOA amoA genes International, Wageningen, The Netherlands). (1.02 × 10 copies/ng DNA). The abundance of AOB amoA genes ranged between 0.54 copies/ng DNA and 1.32 × 10 copies/ng DNA, with the highest amount of AOB amoA genes Results found in the EAS sample. However, the abundance of AOA amoA genes in the two filter samples (FB and FA) was dis- Characteristics of the samples tinctly higher than that of AOB amoA genes. The AOA/AOB ratio in the FB and FA samples was 213.63 and 27.45, The water quality indexes of the collected samples are shown respectively. in Table 1. The concentrations of COD, NH -N and SS Since the abundance of AOA amoA genes in the FB sample sharply decreased following treatment in the primary settling was the highest, we investigated further the distribution of tank and BAF. The temperature variations among the different AOA in the filter layer of the BAF. The characteristics of units in the treatment system were negligible, with the tem- wastewater samples collected at different heights (0.5-m inter- perature ranging between 27.6 and 28.3 °C. However, the DO vals) of the filter layer were measured. As shown in Fig. 3,the concentrations of the five samples fluctuated greatly, with the recorded temperatures at the different heights were in the highest and lowest DO concentration recorded as 4.57 mg O / range of 28.4 to 29.1 °C. The highest pH value was 7.48, L in the EB sample and 0.67 mg O /L in the EA sample, which was recorded at a height of 2.0 m. The pH value de- respectively. The pH value ranged between 6.57 and 7.51. creased to pH 6.66 at a height of 4.0 m, but there was no significant change in pH value from 4 to 5 m in height. Abundance of AOA and AOB in the collected samples However, the concentration of DO increased from 2.12 to 5.78 mg O /L along the direction of filtration. As shown in Figure 2 shows the qPCR results of AOA and AOB amoA Fig. 4a, the NH -N concentration also significantly decreased genes of the collected samples. In general, the qPCR results from 11.98 mg N/L at a height of 2.0 to 2.57 mg N/L at 5 m. In indicated that AOB amoA genes were ubiquitous in the comparison, the NO -N concentration increased at different Table 1 Water quality indexes of the five water samples collected from the wastewater treatment plant Water samples F value P-value Physiochemical properties RW IB EB EA EAS a b c c c COD (mg O /L) 171.25 ± 8.92 48.05 ± 3.54 18.07 ± 3.20 18.75 ± 1.34 18.90 ± 4.37 533.2 0.0000 a ab bc de ce TN (mgN/L) 21.01 ± 2.71 15.50 ± 3.23 13.24 ± 2.68 4.96 ± 0.96 9.06 ± 1.24 20.6 0.0001 − a a b a a NO -N (mg N/L) 0.71 ± 0.23 2.73 ± 0.70 8.96 ± 2.27 2.30 ± 0.67 3.56 ± 1.21 19.5 0.0001 − a a a b a NO -N (mg N/L) 0.30 ± 0.05 0.50 ± 0.26 0.14 ± 0.06 3.42 ± 0.96 0.72 ± 0.42 23.6 0.0000 + a b c c c NH -N (mg N/L) 16.21 ± 2.82 10.61 ± 2.43 3.29 ± 0.68 3.86 ± 0.92 3.13 ± 1.14 30.9 0.0000 a b c c c SS (mg/L) 90 ± 17.35 44 ± 13.53 10 ± 2.65 7 ± 3.00 4 ± 3.46 38.3 0.0000 a b c c c pH 6.99 ± 0.12 7.51 ± 0.05 6.57 ± 0.03 6.73 ± 0.01 6.60 ± 0.03 126.5 0.0000 ab a c a bc DO (mg/L) 1.42 ± 0.22 0.94 ± 0.36 4.57 ± 0.89 0.67 ± 0.03 3.37 ± 1.36 14.8 0.0003 a a a a a Temperature (°C) 28.2 ± 0.10 27.6 ± 0.17 28.0 ± 0.06 27.9 ± 0.10 27.8 ± 0.82 1.1 0.4203 Values in the same row followed by the same lowcase letter are not significantly different at P ≤ 0.05 according to one-way analysis of variance followed by the post hoc Tukey tes) RW, Raw wastewater; IB, influent of the biological aeration filter; EB, effluent of the biological aerated filter (BAF); EA, effluent of the anaerobicfilter; EAS, effluent of ACTIFLO® setting tank b − − + COD, Chemical oxygen demand; TN, total nitrogen; NO -N, nitrate nitrogen; NO -N, nitrite nitrogen; NH -N, ammonium nitrogen; SS, soluble 3 2 4 solids; DO, dissolved oxygen DO concentration (mg/L) Ann Microbiol (2017) 67:405–416 409 - + - TN NO-N NH -N NO -N 3 4 2 AOA a) AOB 0.5 4 12 0.4 3 0.3 0.2 0.1 0.0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Height (m) RW IB EB EA EAS FB FA Samples COD b) Fig. 2 Quantitative analysis of ammonia-oxidizing archaea (AOA)and ammonia-oxidizing bacteria (AOB) amoA genes (encoding ammonia monooxygenase) in the collected samples. RW Raw wastewater, IB influent of the BAF, EB effluent of the BAF, EA effluent of anaerobic filter, EAS effluent of ACTIFLO® settling tank, FB samples taken from the filter layers of the BAF, FA samples taken from the anaerobic filter heights, with minimum and maximum concentrations of 3.64 and 12.07 mg N/L at heights of 2.0 and 5.0 m, respectively. Based on the low variations in TN and NO -N concentrations 2.0 2.5 3.0 3.5 4.0 4.5 5.0 at different heights, we deduced that NH -N was converted to Height (m) NO -N and that the nitrification reactions mainly occurred in Fig. 4 Water quality indexes of the effluent of filter samples collected at the filter layers. The occurrence of nitrification reactions in the different heights of the BAF. a Concentration of total nitrogen (TN), filter layer could also provide a possible explanation for the + − ammonium nitrogen (NH -N), nitrite nitrogen (NO -N) and nitrate 4 2 decrease in pH value. The COD concentration decreased nitrogen (NO -N) b Concentration of chemical oxygen demand (COD) sharply from 161.02 to 46.85 mg/L with increasing height of the BAF filter layers to 5 m (Fig. 4b). at a height of 5.0 m of the BAF. In comparison, the amount of The abundances of AOA and AOB in the filter samples at AOB amoA genes found in the BAF varied between 20.64 and different heights of the BAF are shown in Fig. 5. The abun- 1.05 × 10 copies/ng DNA, with the maximum values record- dance of AOA amoA genes varied between 6.32 × 10 and ed at a height of 4.0 m of the BAF. The ratios of AOA/AOB 3.8 × 10 copies/ng DNA, while maximum value was attained ranged between 845.37 and 2784.91, which indicated that AOAwere dominant over AOB in the BAF. The water quality clearly improved with increasing height along the BAF. Temperture pH DO concentration Diversity of AOA communities The distribution and relative abundance of AOA amoA genes in the WWTP was investigated. Based on AOA amoA gene sequences, we constructed and evaluated five clone libraries and 27 OTUs. The distribution of the relative abundances of OTUs in the collected samples are shown in Fig 6.OTU4 and 22 OTU8 were dominant in the RW and EAS samples, suggest- ing that the AOA communities in the influent and effluent of the WWTP changed little. OTU3 was dominant in FB and FA, 20 1 2.0 2.5 3.0 3.5 4.0 4.5 5.0 which implied that the BAF and anaerobic filter shared the Height (m) same dominant OTU type. OTU3 was also the most widely Fig. 3 Changes in the temperature, pH and dissolved oxygen (DO) distributed OTU type, being present in all five samples tested. concentration of the effluent of filter samples collected at different heights of the BAF The results suggested that OTU3 had a strong adaptability to Temperature (ºC) lg(amoA genes copies) pH COD concentration (mg O /L) TN concentration (mg N/L) AOA/AOB 410 Ann Microbiol (2017) 67:405–416 AOA AOB AOA/AOB Fig. 5 The abundance of AOA 120 3000 and AOB amoA genes in filter 40,000 samples collected at different heights of the BAF 35,000 30,000 25,000 60 1500 20,000 15,000 10,000 5,000 0 0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Height (m) the environment. Although OTU4 dominated in the RW sam- all higher than the samples from the filers (FB and FA), indi- ple, it was not present in the FB sample, suggesting that the cating that the AOA from wastewater had a richer species environmental conditions in the BAF might be not suitable for diversity. The highest H and S values were 2.47 and 34.5, the growth of OTU4. The C, J, H and S values of the AOA respectively, detected from the EAS sample (Table 2). amoA gene clone libraries are presented in Table 2. The results The distribution and relative abundance of AOA amoA showed that RW and EAS contained 12 and 10 OTU types, genes in the filter layer of the BAF was further investigated respectively. In contrast, only five OTU types were detected in (Fig. 7). Six clone libraries were constructed and eight OTUs the FA sample. The FA sample had the minimum H value were obtained from the AOA amoA gene sequences at the 3% (0.54) among the five samples measured, suggesting that the nucleotide cut-off. The relative abundance of OTUs in the diversity of AOA amoA genes in the filter layer of the anaer- samples collected from the heights of 2.0 and 3.5 m was over obic filter was lower than in the other samples. The C value of 60%, and OTUb and OTUc were the dominant types in sam- the FB and FA samples was 90.0 and 90.6%, respectively, ples collected between the heights of 4.0 and 5.0 m. The which indicated that clone libraries covered the AOA commu- relative abundance of both kinds of OTU types was around nities in FA and FB samples and that the majority of species 40%. OTUb was the most prevalent OTU type among the could be detected and identified. For the samples taken from eight OTUs in the BAF, being present in all samples wastewater (RW, EB and EAS), the H value and S value were (Fig. 7). The value of C ranged between 78.6 and 100%, OTU27 100% Fig. 6 Distribution and relative OTU26 abundance of the AOA amoA OTU25 gene in the collected samples. 90% OTU24 OTU Operational taxonomic unit OYU23 OTU22 80% OTU21 OTU20 OTU19 70% OTU18 OTU17 60% OTU16 OTU15 OTU14 50% OTU13 OTU12 40% OTU11 OTU10 OTU9 30% OTU8 OTU7 OTU6 20% OTU5 OTU4 10% OTU3 OTU2 OTU1 0% RW EB FB FA EAS Samples AOB amoA genes (copies/ng DNA) Percentage AOA amoA genes (copies/ng DNA) Ann Microbiol (2017) 67:405–416 411 Table 2 Diversity of each clone library of ammonia oxidizing archaea Table 3 Diversity of each clone library of ammonia oxidizing archaea amoA genes in the five water samples amoA genes at different heights of the biological aerated filter Samples No. of clones OTUs CJ H S Height (m) No. of clones OTUsCJ H S RW 37 12 78.4% 0.65 1.63 21.78 2 9 2 100% 6.770.642.00 EB 18 8 68.2% 0.85 1.95 11.50 2.5 23 3 100% 0.69 0.75 3.00 FB 29 7 90.0% 0.67 1.30 8.88 3 14 4 78.6% 0.540.758.5 FA 32 5 90.6% 0.34 0.54 6.88 3.5 24 4 95.8% 0.69 0.96 4.50 EAS 22 10 77.8% 1.19 2.47 34.5 4 7 3 85.7% 0.91 1.00 3.50 5 28 6 92.8% 0.751.356.11 OTU, Operational taxonomic unit; C, library coverage; H, Shannon– Weiner diversity index; J,Pielou’s evenness index; S Chao 1 richness estimate phylogenetically within the Nitrosopumilus cluster (Jung et al. 2011). OTU8, another dominant OTU type in the RW suggesting that AOA communities covered the whole clone and EAS samples, correlated with sequences collected from libraries of these samples (Table 3). However, the H value the Mississippi River. OTU10 in the EAS sample was seen to increased gradually from 0.64to1.35withincreasingof be closely related to the sequences from WWTPs, which also height from 2.0 to 5.0 m, indicating that the increase of belong to the Nitrosopumilus cluster (Bai et al. 2012; Gao AOA diversity was related to the height of filter layer in BAF. et al. 2014). OTU3, which was dominant in the filter layer of BAF, showed a similarity to the AOA species detected in bench-scale airlift reactor for wastewater treatment. OTU26 Phylogenetic analysis of AOA was strongly correlated with AOA sequences collected from The phylogenetic tree of AOA amoA genes in the municipal rice soil. OTU14 from the FB sample showed a high similarity to sequence WG6612 from Yellowstone hot spring (Zhang WWTPs is shown in Fig. 8. Based on the results, the obtained AOA amoA sequences could be grouped into two major clus- et al. 2008). OTU1 was present in the EB, FB and FA samples and was correlated to SL-37 from the Stanley WWTP that ters. Most of the OTUs in one of the major clusters were strongly correlated with sequences from the natural environ- falls into the Nitrososphaera sister subcluster (Zhang et al. 2009). ment, such as rivers, lakes, estuary sediments and soils. The OTUs in the second major cluster had many similarities to the Eight OTUs also fell into two major clusters, as shown in the AOA phylogenetic tree of the BAF (Fig. 9). OTUd, which sequences from WWTPs. The most abundant OTU type in the RW and EAS samples, OTU4, showed high similarities to the was the dominant OTU type in the BAF filter between the sequence HZNAOA7 from Dongjiang River sediment, which height at 2.0 and 3.5 m, was strongly correlated with the se- quences belonging to sediments of Dongjiang River. OTUb belongs to Nitrosopumilus cluster (Sun et al. 2013). OTU27 from the EB sample was related to the sequences of and OTUc, the dominant types in the BAF between 4.0 and 5.0 m, were similar to the AOA clones detected in a Nitrosopumilaceae archaeon MY1 which fall 100% Fig. 7 Distribution and relative abundance of AOA amoA gene of 90% the filter sample at different OTUh heights of the BAF 80% OTUg OTUf 70% OTUe 60% OTUd 50% OTUc 40% OTUb OTUa 30% 20% 10% 0% 2 2.5 3 3.5 4 5 Height (m) Precentage 412 Ann Microbiol (2017) 67:405–416 Fig. 8 Phylogenetic tree of AOA amoA gene sequence from different samples in the municipal wastewater treatment plants Ann Microbiol (2017) 67:405–416 413 Fig. 9 Phylogenetic tree of AOA amoA gene sequences from different samples in the filter layer of the BAF wastewater treatment bioreactor operating at 30 °C for Discussion 10 days. Most OTUs obtained from the filter layer of BAF fell into the Nitrosocomicus cluster. These results led us to A number of studies have demonstrated the presence of AOA conclude that the AOA near the inlet of the BAF were similar amoA genes in WWTPs, but the abundances of AOA and to the ones from natural environments, while the AOA near AOB amoA genes have remained a focus of controversy. the outlet of BAF were similar to the ones from wastewater AOB amoA genes have been reported to be the dominant type treatment systems. in WWTPs (Wells et al. 2009; Jin et al. 2010;Tongetal. 2011; Bai et al. 2012; Gao et al. 2013). Yapsakli et al. (2011)inves- tigated AOA and AOB abundance in a full-scale leachate Correlations between AOA community structure and environmental factors We used RDA to evaluate the correlations between the AOA community structures in the BAF and environmental factors. The results are shown in Fig. 10, where Axis1 and Axis2 correspond to 73.0 and 10.9% of the cumulative variance of the AOA community–environment relationship, respectively. The location of the red arrows of COD, pH and DO near Axis1 suggest that these factors had significant effects on the community structures of AOA from the filter layer of the BAF. The AOA near the bottom of filter layer (between the height of 2 and 2.5 m) in the BAF were mainly distributed along pH and DO gradients, indicating that the AOA from the bottom of the filter layer were strongly correlated with pH and DO concentrations. The communities of AOA at the top of the filter layer (at the height of 5 m) in the BAF were significantly + − affected by COD, NH -N and NO -N. In addition, the DO 4 2 concentration showed a strong negative correlation with the + − + Fig. 10 Ordination plots generated by redundancy analysis. Red arrows concentrations of COD, NH -N and NO -N. NH -N con- 4 2 4 − − Environmental variables (pH, DO, COD, TN, NO -N, NO -N, and 2 3 centration also showed a strong negative correlation with NH -N), circles and numbers filter samples collected at different heights from the BAF NO -N concentration. 3 414 Ann Microbiol (2017) 67:405–416 treatment plant with high concentration of COD (5000 mg O / wastewater. It has been suggested that some types of AOA L) and TN (2000 mg N/L) and found that AOB were dominant may only be able to survive under low DO concentrations; in the treatment plant, with an AOB/AOA ratio range of one to thus low DO concentrations may be a crucial factor for the three orders of magnitude. These results were confirmed by presence of AOA (Erguder et al. 2009). However, the results Gao et al. (2014) who reported that AOB amoA genes obtained in our study are no in agreement with this specula- outnumbered AOA in ten WWTPs. In contrast, Kayee et al. tion. In our study, the amount of AOA amoA genes increased (2011)found that AOA amoA genes outnumbered AOB amoA with increasing DO concentration in the filter layer. One pos- genes in eight municipal WWTPs, and Bai et al. (2012)dem- sible explanation for this phenomenon is the possibility that onstrated that AOA were dominant in three municipal the AOA types in the BAF were able to adapt to the environ- WWTPs, while AOB were dominant in three industrial ment of a relatively high DO level. It is also possible that the WWTPs. A relatively higher amount of AOA amoA genes oxygen was consumed quickly by heterotrophic microorgan- was also found by Muβmann et al. (2011)aswellas isms on the surface of biofilm and that AOA may be present in Sonthiphand and Limpiyakorn (2011). In the present study, the depths of biofilm at low DO levels. In addition, with the we investigated a full-scale municipal WWTP with a BAF increase in the height of the BAF from 2.0 to 5.0 m, COD and as the main processing unit. The WWTP had a high capacity NH -N concentrations decreased while at the same time there for treating pollutants, with the concentrations of COD and SS was a significant increase of AOA amoA genes. This phenom- in wastewater decreasing from 171.25 mg O /L and 90 mg/L enon suggests that low COD and NH -N levels may be fa- 2 4 to 18.90 mg O /L and 4 mg/L, respectively (Table 1). AOA vorable for the growth of AOA in BAF. DO concentrations and AOB amoA genes were detected in the collected samples and pH values, which changed regularly along the direction of from different sections of the WWTP, and our results showed filtration, may also be important influencing factors. that AOA were dominant in the BAF processing unit. Across AOA have been divided into five major clusters based on the flow path of the treatment system, the abundance of AOA the published AOA amoA gene sequences (Pester et al. 2012): amoA genes in wastewater increased from 26.00 to 217.27 Nitosopumilis cluster, Nitrososphaera cluster, Nitrosocaldus copies/ng DNA, and the NH -N concentration decreased cluster, Nitrosotalea cluster and Nitrosophaera sister cluster. from 16.21 to 3.13 mg N/L (Table 1). The abundance of In the present study, five clone libraries of AOA were con- AOB amoA genes in the wastewater also increased from structed with samples from the WWTP. The obtained AOA 4.18 to 133.97 copies/ng DNA during the process. amoA gene sequences fell into two major clusters. OTU4 and AOA abundance showed a strong, positive association OTU8, which were dominant in the wastewater samples, with temperature and NO -N concentration and a significant, grouped into Cluster A, showing a highly similarity with se- + − negative correlation with NH -N and NO -N concentra- quences from natural environments, which belonged to the 4 2 tions, respectively, and pH value (Table 4). Since NO -N Nitrosopumilus cluster. OTU3 from FB fell into Cluster B, was mainly converted from NH -N oxidation in the BAF, with most sequences obtained from WWTPs. Many studies NO -N concentrations increased concomitantly with the de- have found that most AOA obtained from WWTPs belong to + − crease in NH -N concentrations. In addition, NO -N con- the Nitrososphaera cluster (Muβmann et al. 2011;Tongetal. 4 2 centrations were always very low, at <0.4 mg N/L. The pH 2011; Yapsakli et al. 2011; Sauder et al. 2012; Gao et al. value and NH -N concentration decreased with increasing 2013), but other studies have reported that Nitrosopumilus height of the filter, with a simultaneous increase in AOA and cluster is dominant in WWTPs. A novel AOA strain SAT1 AOB amoA genes. We therefore surmised that NH -N con- enriched from activated sludge has been affiliated with the centration and pH value were the main critical influences on Nitrosopumilus cluster (Li et al. 2016). Similar results were the abundance of AOA. In terms of AOB abundance, the pH also reported by Wu et al. (2013). value and NH -N concentration had a significant negative The abundance and diversity of AOA amoA genes can be influence. As such, AOA and AOB amoA genes shared com- significantly influenced by environmental factors. The in- mon influencing factors. As an electron acceptor, DO is also crease in DO concentrations could promote the degradation considered to be an important factor in the processing of of organic pollutants, and the nitrification process in which Table 4 Statistical analysis of physicochemical parameters and the abundance of amoA genes of AOA and AOB + − − Parameters NH -N NO -N NO -N TN COD DO pH Temperature 4 2 3 AOA amoA genes −0.685** −0.623** 0.536** −0.116 −0.252 0.355 −0.555** 0.596** AOB amoA genes −0.450* −0.316 0.166 −0.387 −0.078 0.380* −0.572** 0.226 **Highly significant at P ≤ 0.01; *significant at P ≤ 0.05 Ann Microbiol (2017) 67:405–416 415 + − − high carbon to nitrogen ratios under micro-aerobic condition. NH -N and NO -N is converted to NO -N correlated neg- 4 2 3 Biores Technol 232:417–422 + − atively to the concentrations of COD, NH -N and NO -N. 4 2 Erguder TH, Boon N, Wittebolle L, Marzorati M, Verstraete W (2009) + − The negative correlation between NH -N and NO -N may 4 3 Environmental factors shaping the ecological niches of ammonia- + − be attributed to the conversion of NH -N into NO -N in the oxidizing archaea. FEMS Microbiol Rev 33(5):855–869 4 3 Ferrera I, Sánchez O (2016) Insights into microbial diversity in wastewa- BAF. The minimal angles between DO, pH, COD and Axis 1 ter treatment systems: how far have we come? Biotechnol Adv 34: also confirmed that the AOA community structure in BAF 790–802 was significantly influenced by DO concentrations, pH value Gao J, Luo X, Wu G, Li T, Peng Y (2014) Abundance and diversity based and COD concentrations (Fig. 10). on amoA genes of ammonia-oxidizing archaea and bacteria in ten wastewater treatment systems. Appl Microbiol Biotechnol 98:3339– In summary, in this study, we investigated the abundance and diversity of AOA in a full-scale WWTP with BAF as the Gao JF, Luo X, Wu GX, Li T, Peng YZ (2013) Quantitative analyses of main processing unit. We found that AOA amoA genes the composition and abundance of ammonia-oxidizing archaea and outnumbered AOB amoA genes in the WWTP, especially in ammonia-oxidizing bacteria in eight full-scale biological wastewater the filter layer of the BAF. The abundance of AOA amoA treatment plants. Bioresour Technol 138C:285–296 3 4 Good IJ (1953) The population frequencies of species and the estimation genes in the BAF varied from 6.32 × 10 to 3.8 × 10 of population parameters. Biometrika 40:237–264 copies/ng DNA, while the highest abundance of AOB amoA Hill TC, Walsh KA, Harris JA, Moffett BF (2003) Using ecological genes was 1.05 × 10 copies/ng DNA. AOAwere dominant in diversity measures with bacterial communities. FEMS Microbiol the BAF, suggesting the important role of this type of micro- Ecol 43:1–11 Hou J, Song C, Cao X, Zhou Y (2013) Shifts between ammonia- organisms on nitrogen removal. The AOA sequences obtained oxidizing bacteria and archaea in relation to nitrification potential from the WWTP were grouped into two major clusters, with across trophic gradients in two large Chinese lakes (Lake Taihu and one cluster strongly correlating with AOA from natural envi- Lake Chaohu). Water Res 47:2285–2296 ronments and the other cluster showing a high similarity to the Jin T, Zhang T, Yan Q (2010) Characterization and quantification of AOA from WWTPs. The AOA in the WWTP were mostly ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR. Appl related to the Nitrosopumilus cluster. pH and NH -N were Microbiol Biotechnol 87:1167–1176 critical environmental factors with a major effect on the abun- Jung MY, Park SJ, Min D, Kim JS, Rijpstra WIC, Damsté JSS, Kim GJ, dance of AOA and AOB amoA genes. AOA community struc- Madsen EL, Rhee SK (2011) Enrichment and characterization of an ture was significantly influenced by DO concentrations, pH autotrophic ammonia-oxidizing archaeon of mesophilic Crenarchaeal group I.1a from an agricultural soil. Appl Environ value and COD concentration. Microbiol 77:8635–8647 Kayee P, Sonthiphand P, Rongsayamanont C, Limpiyakorn T (2011) Acknowledgments This work was supported by the National Natural Archaeal amoA genes outnumber bacterial amoA genes in munici- Science Foundation of China (No.50978069). pal wastewater treatment plants in Bangkok. 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Annals of MicrobiologySpringer Journals

Published: May 26, 2017

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