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Heterogeneity of ammonia-oxidizing community structures in a pilot-scale drinking water biofilter
International Biodeterioration & Biodegradation 70 (2012) 148e152
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Heterogeneity of ammonia-oxidizing community structures in a pilot-scale drinking water biofilter Shuo Feng a,1, Xiaojian Zhang a,1, Qingfeng Wang b, Rui Wan b, Chao Chen a, Shuguang Xie b, * a b
School of Environment, Tsinghua University, Beijing 100084, China College of Environmental Sciences and Engineering, The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2011 Received in revised form 4 March 2012 Accepted 12 March 2012 Available online 6 April 2012
Drinking water biofilters have been widely used for ammonia removal. Knowledge about the structure of ammonia oxidizing communities can aid in understanding of nitrification process. Terminal restriction fragment length polymorphism (TRFLP) analysis of amoA genes in combination with cloning and sequencing analysis were used to investigate spatial heterogeneity of ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) communities in a pilot-scale granular activated carbon (GAC)sand dual media filter. The results illustrate the diversity of AOB communities on GAC samples and their changes along the filter depth. Moreover, Nitrosomonas-like microorganisms were the dominant AOB species in GAC samples. However, AOA was not detected in the biofilter. This work could add some new insights into the nitrification in drinking water biofilters. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Ammonia oxidizing archaea (AOA) Ammonia oxidizing bacteria (AOB) Drinking water Biofilter Nitrosomonas
1. Introduction Ammonia is usually present in source water used for drinking water, especially in regions with intense anthropogenic activities. The presence of ammonia in drinking water might lead to several water quality problems during distribution (Kihn et al., 2000; van der Wielen et al., 2009). Ammonia can be effectively removed through a two-step nitrification process in which sequential oxidation of ammonia into nitrite and then nitrate occurs (Leemann et al., 2010). Nitrosomonas and Nitrobacter are the most common genera, known respectively as ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). However, the discovery of ammonia oxidizing archaea (AOA) has greatly changed our understanding of nitrification (Konneke et al., 2005). Recently, AOA have been found in ground water treatment processes in Japan and the Netherlands (de Vet et al., 2009; van der Wielen et al., 2009; Kasuga et al., 2010). These works suggested that AOA could be responsible for the removal of ammonia in groundwater treatment plants.
* Corresponding author. Tel./fax: þ86 10 62751923. E-mail address: [email protected] (S. Xie). 1 These authors contributed equally to this study. 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2012.03.003
However, the information on the presence of AOA in surface water treatment processes is still lacking. Numerous works have investigated nitrifying biomass and its activity in drinking water biofilters (Niquette et al., 1998; Kihn et al., 2000; Tränckner et al., 2008). The nitrifying biomass could decrease with increasing depth in the drinking water biofilters, as the substrate concentration in the water decreased (Kihn et al., 2000). Knowledge about the structure of nitrifier community can also aid in understanding of nitrification process. However, the structure of nitrifier community in drinking water biofilters has been rarely addressed (Yapsakli et al., 2010; van den Akker et al., 2011; Wahman et al., 2011). Moreover, to the authors’ knowledge, the spatial heterogeneity of nitrifier community in drinking water biofilters has not been identified in the literature before. Granular activated carbon (GAC)-sand dual media filter could effectively remove ammonium, organic matters and turbidity (Yang et al., 2000). It seems a good option to retrofit a sand filter to a GACsand dual media filter, especially where space is limited for water producers to introduce external advanced treatment units. In the current study, spatial heterogeneity of AOA and AOB community structures in a pilot-scale GAC-sand dual media filter was investigated using terminal restriction fragment length polymorphism (TRFLP) analysis in combination with cloning and sequencing analysis.
S. Feng et al. / International Biodeterioration & Biodegradation 70 (2012) 148e152
2. Materials and methods 2.1. Samples Water and particle samples were collected from a pilot-scale GAC-sand dual media filter in down-flow mode. The filtration column was a Plexiglas cylinder (4-m length and 0.3-m-diameter), equipped with sampling ports for water, GAC, and sand (Fig. 1). From top to down, the column was filled with GAC (1-m height), sand (0.4-m height), and gravel (0.3-m height). With a hydraulic loading of 8 m/h, the pilot filter was fed with the settled water from a drinking water plant treating river water. The treatment train of the drinking water treatment plant consists of coagulationeflocculation, sedimentation, rapid sand filtration, and disinfection. Before this study, the dual media filter had been operated for more than eight months allowing for the maturation of nitrifying biomass. During this study, the pH values, oxygen concentrations, and temperatures of the influent ranged between 7.0 and 7.5, 5.0 and 7.9 mg O2/L, and 25 and 30 C, respectively. The ammonia concentrations were determined according to the standard methods (China Environmental Protection Agency, 2002). 2.2. TRFLP for ammonia monooxygenase A genes of AOA and AOB The particle samples were collected from 0.2, 0.4, 1.0, and 1.2 m depth below the surface of the GAC layer, referred to as Sample A, Sample B, Sample C, and Sample D, respectively. DNA was extracted using the UltraClean DNA extraction kit (Mobio Laboratories, Carlsbad, USA). The ammonia monooxygenase A (amoA) gene has been widely used for the study of ammonia oxidizers (Francis et al., 2005). PCR amplification of amoA gene of AOB was carried out with the forward primer amoA-1F (50 -GGGGTTTCTACTGGTGGT-30 ; 50 end-labeled with carboxyfluorescine) and the reverse primer amoA-2R (50 -CCCCTCKGSAAAGCCTTCTTC-30 ) (Horz et al., 2000;
149
Ying et al., 2010). amoA gene of AOA was amplified using ArchamoAF (50 -STAATGGTCTGGCTTAGACG-30 ; 50 end-labeled with carboxyfluorescine) and Arch-amoAR (50 -GCGGCCATCCATCTGTATGT-30 ) (Francis et al., 2005; Ying et al., 2010). PCR reactions were performed as follows: 95 C for 3 min; 35 cycles of 95 C for 45 s, 53 C (for AOA) or 55 C (for AOB) for 1 min, followed by 72 C for 1 min; and finally 72 C for 7 min (Li et al., 2011a). HaeIII and HhaI were selected to digest PCR products purified with QIA quick PCR purification kit (Qiagen Inc., Germany) and may classify the different clone sequences into unique terminal restriction fragments (Zhang et al., 2011a). The clone sequence was first identified, the in silico cut site (HaeIII digest) of which matched the length of the abundant fragment (HaeIII digest). If the clone restriction enzyme (HhaI) cut site predicted from sequence also matched the observed length of the abundant fragment (HhaI), the taxonomic identity of the abundant fragment (HaeIII digest) could be confirmed (Zhang et al., 2011a). The fragment pattern was detected using an ABI 3730 DNA analyzer (Applied Biosystems, Foster, USA). The relative abundance of each terminal restriction fragment was determined by calculating the ratio of the area of each peak to the total area of all peaks in a given TRFLP profile. The peaks with relative abundance <1% or smaller than 50 bp were excluded from further analysis. Ribotype richness (S) equals to the total number of distinct fragments in a profile. The Shannon diversity index (H) and evenness (E) were calculated according to the standard method (Mills et al., 2003). 2.3. Cloning and sequencing The PCR conditions were the same as the above-mentioned, except that the forward primer was unlabeled. The PCR products were cloned into pMD19-T vector (TaKaRa Co., Japan) following the manufacturer’s instruction. The white colonies were verified by PCR with primers M13 F (50 -TGTAAAACGACGGCCAGT-30 ) and M13 R (50 -AACAGCTATGACCATG-30 ). Clones were sequenced at SinoGenoMax Co., Ltd. (Beijing). Clones sharing 98% identity were grouped into one operational taxonomic unit (OTU) using distancebased OTU and richness program (DOTUR) (Schloss and Handelsman, 2005). The nucleotide sequences were compared with those from the GenBank using BLASTn (http://www.ncbi.nlm. nih.gov). Neighbor-joining trees of the sequences in this study and the reference sequences retrieved from the GenBank were constructed using MEGA Version 4.0 with 1,000 replicates (Tamura et al., 2007). Alignment of the sequences was performed using ClustalW (http://www.ebi.ac.uk/clustalw/). The sequences reported in this study were deposited in the GenBank under accession number JN998582-JN998605. 3. Results
Fig. 1. Schematic of the pilot-scale GAC-sand dual media filter.
Although the influent ammonia concentration varied greatly during the 92-day operation, the GAC-sand dual media filter maintained an effective ammonia removal (Fig. 2). Change of ammonia concentration along the filter depth on day 92 is shown in Fig. 3. The result indicates that ammonia removal mainly occurred in GAC layer, especially in the top 0.6-m, however, negligible reduction of ammonia was observed in sand layer. amoA gene of AOB was successfully amplified for Sample A, B and C, but not Sample D. This was consistent with the negligible reduction of ammonia in sand layer. However, all four samples showed no PCR amplification with the widely used primers for amoA gene of AOA. Fig. 4 illustrates the change of AOB community structure in GAC layer along the filter depth. In Sample A fragment 167 bp (HaeIII) was predominant (with a relative abundance of 71%), and there were no other abundant fragments (less than 5%). In
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S. Feng et al. / International Biodeterioration & Biodegradation 70 (2012) 148e152 Influent
Effluent
1.2
NH4+-N (mg/L)
1.0 0.8 0.6 0.4 0.2 0.0 1
6
12
19
22
27 35 46 Times (day)
51
56
70
77
92
Fig. 2. Influent and effluent ammonia concentrations of the GAC-sand dual media filter.
Sample B and C, although fragment 167 bp (HaeIII) was still the most abundant (respectively 35% and 23%), other abundant fragments 67 bp, 130 bp, 149 bp, and 187 bp were also detected. Table 1 shows the differences of AOB community diversity indices among samples. The AOB community structure in Sample A was much different from that in Sample B and C. The AOB represented by the fragment 167 bp (HaeIII) was determined both by sequencing of cloned genes and by TRFLP analysis of Sample A with another additional digests (HhaI) (Fig. 5). The abundant fragments obtained from both TRFLP restriction enzymes were compared to those obtained from in silico digests to identify the sequence of the fragment 167 bp (HaeIII) (Xie et al., 2010, 2011; Zhang et al., 2011a,b). The same to corresponding in silico cut sites, the TRFLP cut sites were 167 bp (HaeIII) and 119 bp (HhaI), respectively. Fig. 5 also shows that 119 bp (HhaI) was the most abundant fragment in all samples. Of the 24 AOB clones sequenced, fragment 167 bp (HaeIII) had 21matches which could be grouped into one OTU at a 2% difference level. The other 3 clones could be clustered as another OTU. Fig. 6 shows the distant phylogenetic relationship of the representative members from the two AOB OTUs. 4. Discussion The archaeal amoA primers Arch-amoAF and Arch-amoAR have been successfully used to detect AOA in many ecosystems, such as marine water and sediments (Francis et al., 2005; Cao et al., 2011a, c; Li et al., 2011a), deep-ocean (Cao et al., 2011b, d, 2012); soils (Adair and Schwartz, 2008), oil reservoirs (Li et al., 2011b),
1.2
mg/L
0.8
NH4+-N
1.0
0.6
Fig. 4. AOB TRFLP profiles (HaeIII) of GAC samples along the depth.
wastewater treatment processes (Zhang et al., 2009), drinking water distribution systems (van der Wielen et al., 2009), and drinking water biofilters (van der Wielen et al., 2009; Kasuga et al., 2010). van der Wielen et al. (2009) concluded that AOA could be responsible for the removal of ammonia in distribution systems and groundwater treatment processes. Kasuga et al. (2010) suggested that AOA may account for most of the ammonia oxidation on granular activated carbon used in a full-scale advanced drinking water treatment plant. However, this was the first study to investigate the role of AOA in the biofilter used for surface water treatment. Unfortunately, AOA was below detection limit or absent from the GAC and sand samples. Up to date, the isolated chemolithotrophic AOB are confined to beta- or gamma-subclass of Proteobacteria. A recent review indicated that molecular techniques have revealed a wide variety of AOB in many ecosystems (Junier et al., 2010). The diversity of AOB was also found in full-scale drinking water distribution systems (Lipponen et al., 2004). However, the diversity of AOB in drinking water biofilter has still poorly studied. A recent work using cloning and sequencing analysis reported the diversity of AOB community in GAC columns for drinking water treatment (Yapsakli et al., 2010). amoA-based TRFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples (Horz et al., 2000). This was the first work to investigate the spatial heterogeneity of AOB community in drinking water biofilter. amoA-based TRFLP analysis shows the diversity of AOB communities in GAC samples and their changes along the filter depth. The decrease of ammonium concentration along the filter depth might be responsible for change of AOB communities (Limpiyakorn et al., 2007). The higher ammonium concentration favored the abundance of fragment 167 bp (HaeIII), which might be mainly responsible for the removal of ammonia in the GAC-sand duel filter.
0.4 Table 1 Comparison of diversity and evenness indices for the TRFLP profiles (HaeIII digest) from the three GAC samples.
0.2 0.0 0
0.2
0.4
0.6
0.8
1
1.2
1.4
Filter depth m Fig. 3. Change of ammonia concentration along the filter depth. The values on the Xaxis represent the depth below the surface of the GAC layer.
Samples
Ribotype (S)
Diversity (H)
Evenness (E)
A B C
13 22 25
1.75 3.26 3.72
0.53 0.80 0.87
S ¼ total number of bands/profile (richness). H ¼ Shannon diversity index, P H ¼ (pi) (log2pi), where pi is the individual peak area. Hmax ¼ log2 (S). E ¼ H/Hmax.
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species in the GAC layers, implying their important contribution to ammonia removal. Acknowledgments This work was financially supported by Major Science and Technology Program for Water Pollution Control and Treatment (2009ZX07423-003). References
Fig. 5. AOB TRFLP profiles (HhaI) of GAC samples along the depth.
47 57
Nitrosomonas sp. Nm59 (AY123831.1) Nitrosomonas sp. Nm84 (AY123818.1) Nitrosomonas sp. JL21 (AF327919.1) B17 (JN998598) (21) (HaeIII 167 bp) B16 (JN998597) (3)
0.02
Fig. 6. Phylogenetic tree of the representative AOB sequences of each OTU and their reference sequences from GenBank. The obtained sequences beginning with ‘B’ were referred to AOB sequences recovered from Sample A. The bold number in parentheses represents the numbers of the sequences in the same OTU. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analysis of 1,000 resampled datasets. The bar represents 2% sequence divergence.
The cloning and sequencing analysis also shows two distinct clusters of AOB in Sample A. Sequence B17, which was a representative AOB amoA gene sequence with a HaeIII cut site of 167 bp, showed 88% identity to amoA gene sequence (AF327919.1) of a Nitrosomonas species which was obtained from activated sludge (Suwa et al., 1997). Sequence B17 also showed 88% identity to other two amoA gene sequences (AY123831.1 and AY123818.1) of Nitrosomonas species (Purkhold et al., 2003). Sequence B16 showed only 80% identity to sequence B17 and was not closely related to amoA gene sequence of any isolated AOB species reported in GenBank database. Moreover, no Nitrosospira amoA-like sequences were detected in this study, demonstrating that the conditions in the drinking water biofilter favored growth of Nitrosomonas species. The dominance of Nitrosomonas-like microorganisms have also been found in drinking water biolfilters, such as biological activated carbon filter (Yapsakli et al., 2010), and aerated submerged biofilm media of fibrous reactor (Qin et al., 2007). 5. Conclusions AOA was not detected in a pilot-scale GAC-sand dual media filter used for surface water treatment. amoA-based TRFLP analysis shows the diversity of AOB communities in GAC samples and their changes along the filter depth. Nitrosomonas-like microorganisms represented by HaeIII cut site 167 bp, were the most abundant AOB
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Purkhold, U., Wagner, M., Timmermann, G., Pommerening-Roser, A., Koops, H.P., 2003. 16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads. International Journal of Systematic and Evolutionary Microbiology 53, 1485e1494. Qin, Y.Y., Li, D.T., Yang, H., 2007. Investigation of total bacterial and ammoniaoxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China. FEMS Microbiology Letters 268, 126e134. Schloss, P.D., Handelsman, J., 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Applied and Environmental Microbiology 71, 1501e1506. Suwa, Y., Sumino, T., Noto, K., 1997. Phylogenetic relationships of activated sludge isolates of ammonia oxidizers with different sensitivities to ammonium sulfate. The Journal of General and Applied Microbiology 43, 373e379. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4, molecular evolutionary genetics analysis, MEGA software version 4.0. Molecular Biology and Evolution 24, 1596e1599. Tränckner, J., Wricke, B., Krebs, P., 2008. Estimating nitrifying biomass in drinking water filters for surface water treatment. Water Research 42, 2574e2584. van den Akker, B., Holmes, M., Pearce, P., Cromar, N.J., Fallowfield, H.J., 2011. Structure of nitrifying biofilms in a high-rate trickling filter designed for potable water pre-treatment. Water Research 45, 3489e3498. van der Wielen, P.W.J.J., Voost, S., van der Kooij, D., 2009. Ammonia-oxidizing bacteria and archaea in groundwater treatment and drinking water distribution systems. Applied and Environmental Microbiology 75, 4687e4695.
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Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Heterogeneity of ammonia-oxidizing community structures in a pilot-scale drinking water biofilter Shuo Feng a,1, Xiaojian Zhang a,1, Qingfeng Wang b, Rui Wan b, Chao Chen a, Shuguang Xie b, * a b
School of Environment, Tsinghua University, Beijing 100084, China College of Environmental Sciences and Engineering, The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 December 2011 Received in revised form 4 March 2012 Accepted 12 March 2012 Available online 6 April 2012
Drinking water biofilters have been widely used for ammonia removal. Knowledge about the structure of ammonia oxidizing communities can aid in understanding of nitrification process. Terminal restriction fragment length polymorphism (TRFLP) analysis of amoA genes in combination with cloning and sequencing analysis were used to investigate spatial heterogeneity of ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) communities in a pilot-scale granular activated carbon (GAC)sand dual media filter. The results illustrate the diversity of AOB communities on GAC samples and their changes along the filter depth. Moreover, Nitrosomonas-like microorganisms were the dominant AOB species in GAC samples. However, AOA was not detected in the biofilter. This work could add some new insights into the nitrification in drinking water biofilters. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Ammonia oxidizing archaea (AOA) Ammonia oxidizing bacteria (AOB) Drinking water Biofilter Nitrosomonas
1. Introduction Ammonia is usually present in source water used for drinking water, especially in regions with intense anthropogenic activities. The presence of ammonia in drinking water might lead to several water quality problems during distribution (Kihn et al., 2000; van der Wielen et al., 2009). Ammonia can be effectively removed through a two-step nitrification process in which sequential oxidation of ammonia into nitrite and then nitrate occurs (Leemann et al., 2010). Nitrosomonas and Nitrobacter are the most common genera, known respectively as ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). However, the discovery of ammonia oxidizing archaea (AOA) has greatly changed our understanding of nitrification (Konneke et al., 2005). Recently, AOA have been found in ground water treatment processes in Japan and the Netherlands (de Vet et al., 2009; van der Wielen et al., 2009; Kasuga et al., 2010). These works suggested that AOA could be responsible for the removal of ammonia in groundwater treatment plants.
* Corresponding author. Tel./fax: þ86 10 62751923. E-mail address: [email protected] (S. Xie). 1 These authors contributed equally to this study. 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2012.03.003
However, the information on the presence of AOA in surface water treatment processes is still lacking. Numerous works have investigated nitrifying biomass and its activity in drinking water biofilters (Niquette et al., 1998; Kihn et al., 2000; Tränckner et al., 2008). The nitrifying biomass could decrease with increasing depth in the drinking water biofilters, as the substrate concentration in the water decreased (Kihn et al., 2000). Knowledge about the structure of nitrifier community can also aid in understanding of nitrification process. However, the structure of nitrifier community in drinking water biofilters has been rarely addressed (Yapsakli et al., 2010; van den Akker et al., 2011; Wahman et al., 2011). Moreover, to the authors’ knowledge, the spatial heterogeneity of nitrifier community in drinking water biofilters has not been identified in the literature before. Granular activated carbon (GAC)-sand dual media filter could effectively remove ammonium, organic matters and turbidity (Yang et al., 2000). It seems a good option to retrofit a sand filter to a GACsand dual media filter, especially where space is limited for water producers to introduce external advanced treatment units. In the current study, spatial heterogeneity of AOA and AOB community structures in a pilot-scale GAC-sand dual media filter was investigated using terminal restriction fragment length polymorphism (TRFLP) analysis in combination with cloning and sequencing analysis.
S. Feng et al. / International Biodeterioration & Biodegradation 70 (2012) 148e152
2. Materials and methods 2.1. Samples Water and particle samples were collected from a pilot-scale GAC-sand dual media filter in down-flow mode. The filtration column was a Plexiglas cylinder (4-m length and 0.3-m-diameter), equipped with sampling ports for water, GAC, and sand (Fig. 1). From top to down, the column was filled with GAC (1-m height), sand (0.4-m height), and gravel (0.3-m height). With a hydraulic loading of 8 m/h, the pilot filter was fed with the settled water from a drinking water plant treating river water. The treatment train of the drinking water treatment plant consists of coagulationeflocculation, sedimentation, rapid sand filtration, and disinfection. Before this study, the dual media filter had been operated for more than eight months allowing for the maturation of nitrifying biomass. During this study, the pH values, oxygen concentrations, and temperatures of the influent ranged between 7.0 and 7.5, 5.0 and 7.9 mg O2/L, and 25 and 30 C, respectively. The ammonia concentrations were determined according to the standard methods (China Environmental Protection Agency, 2002). 2.2. TRFLP for ammonia monooxygenase A genes of AOA and AOB The particle samples were collected from 0.2, 0.4, 1.0, and 1.2 m depth below the surface of the GAC layer, referred to as Sample A, Sample B, Sample C, and Sample D, respectively. DNA was extracted using the UltraClean DNA extraction kit (Mobio Laboratories, Carlsbad, USA). The ammonia monooxygenase A (amoA) gene has been widely used for the study of ammonia oxidizers (Francis et al., 2005). PCR amplification of amoA gene of AOB was carried out with the forward primer amoA-1F (50 -GGGGTTTCTACTGGTGGT-30 ; 50 end-labeled with carboxyfluorescine) and the reverse primer amoA-2R (50 -CCCCTCKGSAAAGCCTTCTTC-30 ) (Horz et al., 2000;
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Ying et al., 2010). amoA gene of AOA was amplified using ArchamoAF (50 -STAATGGTCTGGCTTAGACG-30 ; 50 end-labeled with carboxyfluorescine) and Arch-amoAR (50 -GCGGCCATCCATCTGTATGT-30 ) (Francis et al., 2005; Ying et al., 2010). PCR reactions were performed as follows: 95 C for 3 min; 35 cycles of 95 C for 45 s, 53 C (for AOA) or 55 C (for AOB) for 1 min, followed by 72 C for 1 min; and finally 72 C for 7 min (Li et al., 2011a). HaeIII and HhaI were selected to digest PCR products purified with QIA quick PCR purification kit (Qiagen Inc., Germany) and may classify the different clone sequences into unique terminal restriction fragments (Zhang et al., 2011a). The clone sequence was first identified, the in silico cut site (HaeIII digest) of which matched the length of the abundant fragment (HaeIII digest). If the clone restriction enzyme (HhaI) cut site predicted from sequence also matched the observed length of the abundant fragment (HhaI), the taxonomic identity of the abundant fragment (HaeIII digest) could be confirmed (Zhang et al., 2011a). The fragment pattern was detected using an ABI 3730 DNA analyzer (Applied Biosystems, Foster, USA). The relative abundance of each terminal restriction fragment was determined by calculating the ratio of the area of each peak to the total area of all peaks in a given TRFLP profile. The peaks with relative abundance <1% or smaller than 50 bp were excluded from further analysis. Ribotype richness (S) equals to the total number of distinct fragments in a profile. The Shannon diversity index (H) and evenness (E) were calculated according to the standard method (Mills et al., 2003). 2.3. Cloning and sequencing The PCR conditions were the same as the above-mentioned, except that the forward primer was unlabeled. The PCR products were cloned into pMD19-T vector (TaKaRa Co., Japan) following the manufacturer’s instruction. The white colonies were verified by PCR with primers M13 F (50 -TGTAAAACGACGGCCAGT-30 ) and M13 R (50 -AACAGCTATGACCATG-30 ). Clones were sequenced at SinoGenoMax Co., Ltd. (Beijing). Clones sharing 98% identity were grouped into one operational taxonomic unit (OTU) using distancebased OTU and richness program (DOTUR) (Schloss and Handelsman, 2005). The nucleotide sequences were compared with those from the GenBank using BLASTn (http://www.ncbi.nlm. nih.gov). Neighbor-joining trees of the sequences in this study and the reference sequences retrieved from the GenBank were constructed using MEGA Version 4.0 with 1,000 replicates (Tamura et al., 2007). Alignment of the sequences was performed using ClustalW (http://www.ebi.ac.uk/clustalw/). The sequences reported in this study were deposited in the GenBank under accession number JN998582-JN998605. 3. Results
Fig. 1. Schematic of the pilot-scale GAC-sand dual media filter.
Although the influent ammonia concentration varied greatly during the 92-day operation, the GAC-sand dual media filter maintained an effective ammonia removal (Fig. 2). Change of ammonia concentration along the filter depth on day 92 is shown in Fig. 3. The result indicates that ammonia removal mainly occurred in GAC layer, especially in the top 0.6-m, however, negligible reduction of ammonia was observed in sand layer. amoA gene of AOB was successfully amplified for Sample A, B and C, but not Sample D. This was consistent with the negligible reduction of ammonia in sand layer. However, all four samples showed no PCR amplification with the widely used primers for amoA gene of AOA. Fig. 4 illustrates the change of AOB community structure in GAC layer along the filter depth. In Sample A fragment 167 bp (HaeIII) was predominant (with a relative abundance of 71%), and there were no other abundant fragments (less than 5%). In
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Effluent
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NH4+-N (mg/L)
1.0 0.8 0.6 0.4 0.2 0.0 1
6
12
19
22
27 35 46 Times (day)
51
56
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77
92
Fig. 2. Influent and effluent ammonia concentrations of the GAC-sand dual media filter.
Sample B and C, although fragment 167 bp (HaeIII) was still the most abundant (respectively 35% and 23%), other abundant fragments 67 bp, 130 bp, 149 bp, and 187 bp were also detected. Table 1 shows the differences of AOB community diversity indices among samples. The AOB community structure in Sample A was much different from that in Sample B and C. The AOB represented by the fragment 167 bp (HaeIII) was determined both by sequencing of cloned genes and by TRFLP analysis of Sample A with another additional digests (HhaI) (Fig. 5). The abundant fragments obtained from both TRFLP restriction enzymes were compared to those obtained from in silico digests to identify the sequence of the fragment 167 bp (HaeIII) (Xie et al., 2010, 2011; Zhang et al., 2011a,b). The same to corresponding in silico cut sites, the TRFLP cut sites were 167 bp (HaeIII) and 119 bp (HhaI), respectively. Fig. 5 also shows that 119 bp (HhaI) was the most abundant fragment in all samples. Of the 24 AOB clones sequenced, fragment 167 bp (HaeIII) had 21matches which could be grouped into one OTU at a 2% difference level. The other 3 clones could be clustered as another OTU. Fig. 6 shows the distant phylogenetic relationship of the representative members from the two AOB OTUs. 4. Discussion The archaeal amoA primers Arch-amoAF and Arch-amoAR have been successfully used to detect AOA in many ecosystems, such as marine water and sediments (Francis et al., 2005; Cao et al., 2011a, c; Li et al., 2011a), deep-ocean (Cao et al., 2011b, d, 2012); soils (Adair and Schwartz, 2008), oil reservoirs (Li et al., 2011b),
1.2
mg/L
0.8
NH4+-N
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0.6
Fig. 4. AOB TRFLP profiles (HaeIII) of GAC samples along the depth.
wastewater treatment processes (Zhang et al., 2009), drinking water distribution systems (van der Wielen et al., 2009), and drinking water biofilters (van der Wielen et al., 2009; Kasuga et al., 2010). van der Wielen et al. (2009) concluded that AOA could be responsible for the removal of ammonia in distribution systems and groundwater treatment processes. Kasuga et al. (2010) suggested that AOA may account for most of the ammonia oxidation on granular activated carbon used in a full-scale advanced drinking water treatment plant. However, this was the first study to investigate the role of AOA in the biofilter used for surface water treatment. Unfortunately, AOA was below detection limit or absent from the GAC and sand samples. Up to date, the isolated chemolithotrophic AOB are confined to beta- or gamma-subclass of Proteobacteria. A recent review indicated that molecular techniques have revealed a wide variety of AOB in many ecosystems (Junier et al., 2010). The diversity of AOB was also found in full-scale drinking water distribution systems (Lipponen et al., 2004). However, the diversity of AOB in drinking water biofilter has still poorly studied. A recent work using cloning and sequencing analysis reported the diversity of AOB community in GAC columns for drinking water treatment (Yapsakli et al., 2010). amoA-based TRFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples (Horz et al., 2000). This was the first work to investigate the spatial heterogeneity of AOB community in drinking water biofilter. amoA-based TRFLP analysis shows the diversity of AOB communities in GAC samples and their changes along the filter depth. The decrease of ammonium concentration along the filter depth might be responsible for change of AOB communities (Limpiyakorn et al., 2007). The higher ammonium concentration favored the abundance of fragment 167 bp (HaeIII), which might be mainly responsible for the removal of ammonia in the GAC-sand duel filter.
0.4 Table 1 Comparison of diversity and evenness indices for the TRFLP profiles (HaeIII digest) from the three GAC samples.
0.2 0.0 0
0.2
0.4
0.6
0.8
1
1.2
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Filter depth m Fig. 3. Change of ammonia concentration along the filter depth. The values on the Xaxis represent the depth below the surface of the GAC layer.
Samples
Ribotype (S)
Diversity (H)
Evenness (E)
A B C
13 22 25
1.75 3.26 3.72
0.53 0.80 0.87
S ¼ total number of bands/profile (richness). H ¼ Shannon diversity index, P H ¼ (pi) (log2pi), where pi is the individual peak area. Hmax ¼ log2 (S). E ¼ H/Hmax.
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species in the GAC layers, implying their important contribution to ammonia removal. Acknowledgments This work was financially supported by Major Science and Technology Program for Water Pollution Control and Treatment (2009ZX07423-003). References
Fig. 5. AOB TRFLP profiles (HhaI) of GAC samples along the depth.
47 57
Nitrosomonas sp. Nm59 (AY123831.1) Nitrosomonas sp. Nm84 (AY123818.1) Nitrosomonas sp. JL21 (AF327919.1) B17 (JN998598) (21) (HaeIII 167 bp) B16 (JN998597) (3)
0.02
Fig. 6. Phylogenetic tree of the representative AOB sequences of each OTU and their reference sequences from GenBank. The obtained sequences beginning with ‘B’ were referred to AOB sequences recovered from Sample A. The bold number in parentheses represents the numbers of the sequences in the same OTU. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analysis of 1,000 resampled datasets. The bar represents 2% sequence divergence.
The cloning and sequencing analysis also shows two distinct clusters of AOB in Sample A. Sequence B17, which was a representative AOB amoA gene sequence with a HaeIII cut site of 167 bp, showed 88% identity to amoA gene sequence (AF327919.1) of a Nitrosomonas species which was obtained from activated sludge (Suwa et al., 1997). Sequence B17 also showed 88% identity to other two amoA gene sequences (AY123831.1 and AY123818.1) of Nitrosomonas species (Purkhold et al., 2003). Sequence B16 showed only 80% identity to sequence B17 and was not closely related to amoA gene sequence of any isolated AOB species reported in GenBank database. Moreover, no Nitrosospira amoA-like sequences were detected in this study, demonstrating that the conditions in the drinking water biofilter favored growth of Nitrosomonas species. The dominance of Nitrosomonas-like microorganisms have also been found in drinking water biolfilters, such as biological activated carbon filter (Yapsakli et al., 2010), and aerated submerged biofilm media of fibrous reactor (Qin et al., 2007). 5. Conclusions AOA was not detected in a pilot-scale GAC-sand dual media filter used for surface water treatment. amoA-based TRFLP analysis shows the diversity of AOB communities in GAC samples and their changes along the filter depth. Nitrosomonas-like microorganisms represented by HaeIII cut site 167 bp, were the most abundant AOB
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