Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6033-8
ENVIRONMENTAL BIOTECHNOLOGY
Effect of inoculum sources on the enrichment of nitrite-dependent anaerobic methane-oxidizing bacteria Zhanfei He & Chen Cai & Lidong Shen & Liping Lou & Ping Zheng & Xinhua Xu & Baolan Hu
Received: 6 July 2014 / Revised: 11 August 2014 / Accepted: 15 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Nitrite-dependent anaerobic methane oxidation (n-damo) is a newly discovered biological process that couples anaerobic oxidation of methane (AOM) to nitrite reduction. In this study, three different inocula, methanogenic sludge, paddy soil, and freshwater sediment were used to enrich n-damo bacteria in three sequencing batch reactors (SBRs), and three n-damo enrichment cultures, C1, C2 and C3, were obtained, respectively. After 500 days of incubation, Methylomirabilis oxyfera-like bacteria and n-damo activities were observed in cultures C1, C2, and C3, and the specific activities were 0.8±0.1, 1.4±0.1, and 1.0±0.1 μmol CH4 h−1 g−1 VSS, respectively. The copy numbers of 16S rRNA genes from cultures C1, C2, and C3 were 5.0±0.4×108, 6.1± 0.1×109, and 1.0±0.2×109 copies g−1 dry weight, respectively. The results indicated that paddy soil is an excellent inoculum for n-damo bacterial enrichment. This work expanded the alternative source of n-damo inoculum and benefited the further research of n-damo process. Keywords n-damo . Inoculum . Paddy soil . Methanogenic sludge . Freshwater sediment
Introduction Anaerobic oxidation of methane (AOM) mediated by microbes is crucial in its role as a sink of the global methane cycle, since it consumes approximately 7–25 % of the annual Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6033-8) contains supplementary material, which is available to authorized users. Z. He : C. Cai : L. Shen : L. Lou : P. Zheng : X. Xu : B. Hu (*) Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China e-mail: [email protected]
global methane production (Knittel and Boetius 2009). At present, investigations of AOM are mainly focused on AOM coupled to sulfate reduction (Knittel and Boetius 2009), but AOM coupled to nitrite reduction (Eq. 1), commonly named nitrite-dependent anaerobic methane oxidation (n-damo), also plays an important role in the methane cycle (Hu et al. 2014b). Raghoebarsing et al. (2006) successfully achieved an enrichment culture capable of AOM coupled to nitrite reduction from anoxic sediment from a canal, which was the first solid evidence for the existence of the n-damo process. The microorganisms responsible for n-damo process were affiliated with the uncultured NC10 phylum (Ettwig et al. 2009) and provisionally named Candidatus “Methylomirabilis oxyfera” (M. oxyfera) (Ettwig et al. 2010). 3CH4 þ 8NO2 − þ 8Hþ → 3CO2 þ 4N2 þ 10H2 O △G0’¼ −928 kJ mol−1 CH4
ð1Þ Hitherto, some n-damo cultures have been enriched successfully (Ettwig et al. 2009; Hu et al. 2009; Kampman et al. 2012, 2014; Luesken et al. 2011b; Raghoebarsing et al. 2006; Zhu et al. 2012), and the proportion of M. oxyfera-like bacteria ranged from 15 to 80 % of the total microorganisms in these cultures. The sources of inocula to enrich for n-damo cultures included freshwater sediment (Ettwig et al. 2009; Kampman et al. 2012; Raghoebarsing et al. 2006); wastewater sludge (Kampman et al. 2014; Luesken et al. 2011b); a mixture of freshwater lake sediment, anaerobic sludge, and return activated sludge (Hu et al. 2009); and minerotrophic peatland soil (Zhu et al. 2012). However, the source of n-damo inoculum was still limited; the effect of inoculum sources was unclear; and highly active n-damo culture was not achieved, which restricted the research of the n-damo process and its biotechnological application. In this study, methanogenic sludge, paddy soil, and freshwater sediment were used as inocula to enrich n-damo bacteria
Appl Microbiol Biotechnol
in three sequencing batch reactors (SBRs), and the obtained cultures were marked as C1, C2, and C3, respectively. Molecular techniques, including quantitative PCR (qPCR), 16S ribosomal RNA (rRNA) gene sequencing, pmoA gene sequencing, and fluorescence in situ hybridization (FISH), were employed to monitor changes in the microbial community before and after the cultivation period. The objective of this study was to determine the feasibility of using the three inocula above for the enrichment of n-damo bacteria, to expand the source of n-damo inocula, and to investigate the effect of inoculum source on the enrichment.
Materials and methods Inocula and medium Methanogenic sludge was sampled from a methanogenic granular sludge reactor; paddy soil was obtained from a paddy field with long-term fertilization (sampling depth 5–10 cm) in Zhejiang Academy of Agricultural Science (30° 18′ 41″ N, 120° 11′ 20″ E), Hangzhou, China; and freshwater sediment was obtained from Tiesha River (30° 15′ 59″ N, 120° 11′ 01″ E; sampling depth, top 20 cm of the sediment) located in the east of Hangzhou, China. The physicochemical characteristics of the three inocula are presented in Table S1. Natural freshwater was taken from the same location where the river sediment was sampled. This kind of natural freshwater with additive nitrite was used as the medium to enrich n-damo bacteria. During the 500 days of enrichment period, the influent concentration of nitrite raised from 0.5 to 1.5 mmol L−1; the influent pH was kept at 7.0–7.2; and the methane partial pressure in headspace was about 20 kPa.
Enrichment protocol Three identical SBRs were used to enrich n-damo bacteria (Fig. S1). The volume of each reactor was 2.5 L, consisted of 0.5 L of inoculum, 1.5 L of medium, and 0.5 L of headspace. Each reactor was mixed by a magnetic stirrer at 150 rpm and operated at 30±1 °C. The reactors were operated in sequencing batch mode with 72 h per cycle. Each cycle consisted of 0.5 h of medium supply, 70 h of incubation, 1 h of biomass settling, and 0.5 h of supernatant liquid discharge. During the medium supply period, pure Ar (99.999 %) was sparged into the reactors to maintain anaerobic condition. Then, 100 mL of methane (purity 99.99 %) was injected into the gas phase of each SBR. After settling, 1 L of supernatant was replaced by same volume of fresh medium.
Activity measurement The n-damo activities of cultures C1, C2, and C3 were measured by batch tests after 500 days of enrichment. Cultures sampled from the reactors were rinsed with nitrite-free medium three times, and then 10 mL of each settled culture was transferred into 72-mL serum bottles. Medium with 0.2 mmol L−1 of nitrite was added to reach a working volume of 50 mL. Subsequently, the serum bottles were flushed with pure Ar (99.999 %) for 15 min, sealed with stoppers, and added with 0.25 mL of pure methane (99.99 %). Assays were performed in a shaking table at 100 rpm and 30±0.5 °C. After 2 h of pre-incubation, 20 μL gas samples and 1 mL liquid samples were taken every 2 h from each bottle to analyze the concentrations of methane (in triplicate), nitrite, and nitrate during the next 10 h. After batch tests, volatile suspended solids (VSS) was measured to characterize the biomass of the cultures. DNA extraction and PCR amplification Samples (0.25 g) of each enrichment culture were collected. DNA extraction of each sample was performed as described previously (Hu et al. 2014a). The primers (Table S2) and amplification conditions used in this study were previously described by Luesken et al. (2011c). The PCR products were evaluated by electrophoresis in a 1.0 % agarose gel. Cloning and sequencing The PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Competent cells transformed with vectors were shaken in SOC medium for 1 h and then grown in LB medium containing ampicillin, X-Gal, and IPTG for 16 h at 37 °C. Clones with successful ligation were detected using the blue-white screening technique. Approximately 20 positive clones from each sample were sequenced by BGI-Shanghai (China). Phylogenetic analysis of 16S rRNA genes and pmoA genes Phylogenetic analysis of the obtained sequences was performed using Mega 4.0 (Tamura et al. 2007). The 16S rRNA and pmoA gene sequences were aligned with the ClustalW algorithm. Phylogenetic trees were constructed by the neighbor-joining method with p-distance correction, and the bootstrap values were based on 1,000 replicates. Operational taxonomic units (OTUs) for the determination of 16S rRNA and pmoA genes diversity were analyzed using 3 and 7 % differences in the nucleotide sequences (Lüke and Frenzel 2011), respectively, as determined by the DOTUR program (Schloss and Handelsman 2005). The
Appl Microbiol Biotechnol
The representative sequences reported in this study have been deposited in the GenBank database under accession numbers KC935359–KC935361 (16S rRNA) and KC935362– KC935371 (pmoA). Quantitative PCR analysis qPCR was performed with an iCycler iQ5 thermocycler and a real-time detection system (Bio-Rad, CA, USA) using specific primer set qP1F/qP1R (Ettwig et al. 2009). Thermal cycling program with annealing temperature of 63 °C were performed as previously described (Ettwig et al. 2009). The construction of the standard curve was conducted from a series of tenfold dilutions of plasmid DNA inserted by 16S rRNA genes of NC10 bacteria amplified through qPCR. Three replicates of each sample and plasmid DNA were conducted. The copy numbers of the samples were determined through a calculation based on comparison with the threshold cycle values of the standard curve. FISH
The enrichment period could be divided into three phases according to the nitrite concentration of the influent (Fig. 1). The nitrite concentration in the influent was 0.5, 1.0, and 1.5 mmol L−1 in phase I (0–180 days), phase II (181–400 days), and phase III (401–500 days), respectively. In phase I, the nitrite-reducing activity of culture C1 remained relatively constant, with an average value of 0.17 mmol L−1 day−1. In contrast, the nitrite-reducing activities of cultures C2 and C3 gradually decreased to about 0.05 and 0.09 mmol L−1 day−1, respectively. In phase II, the nitrite-reducing activity of culture C1 rapidly increased to 0.26 mmol L−1 day−1, mainly caused by the increase of influence nitrite that improved the biological activities. In contrast, during days 181–280, the nitritereducing activities of cultures C2 and C3 were at a low level (about 0.07 mmol L−1 day−1) but sharply increased to 0.20 and 0.23 mmol L−1 day−1, respectively, after 280 days. In phase III, the nitrite-reducing activities of cultures C1, C2, and C3 increased, and the average nitrite-reducing activities were 0.33, 0.32, and 0.34 mmol L−1 day−1, respectively. The doubling time of n-damo bacteria in the cultures were estimated based on the rapid increase in the nitrite-reducing activities using the method reported by He et al. (2013). The doubling times in cultures C1, C2, and C3 were estimated to be 3.2, 2.1, and 3.0 months from days 330–430, 330–430, and 250–430, respectively. n-damo activities of cultures The results of batch activity tests indicated that methane and nitrite could be simultaneously utilized by cultures C1, C2, 0.5
Phase I
Analytical methods The concentrations of ammonium, nitrite, and nitrate were measured using phenate method, colorimetric method, and ultraviolet spectrophotometric screening method (APHA 2005), respectively, and VSS levels were also determined according to the APHA standard methods (APHA 2005). Methane was quantified using an Agilent 6890 gas chromatograph (Agilent, USA) as described previously (Liebner and Wagner 2007). Transmission electron micrograph (TEM) was utilized as described in previous literatures (Jin et al. 2007, 2014). Statistical analysis was performed using IBM SPSS Statistics® Desktop version 19.0 (IBM, USA).
phase II
phase III
0.4
C1 C2 C3
-1
Samples of cultures C1, C2, and C3 were fixed and hybridized after 500 days of enrichment. Formamide (40 %) was combined with the following oligonucleotide probes: S-*DBACT-1027-a-A-18 (red), which is specific for dominant bacteria affiliated with the NC10 phylum (Raghoebarsing et al. 2006), and a mixture of EUB I-III (blue), which detects most bacteria (Daims et al. 1999). FISH images were obtained using a two-photon laser confocal microscope (Zeiss, LSM710 NLO, Germany).
Enrichment of n-damo bacteria
-1
Nucleotide sequence accession numbers
Results
nitrite consumption rate (mmol L d )
equation C=[1−(n1 /N)]×100 % was employed to determine the cloning coverage of each sample (n1 is the number of OTUs containing only one sequence; N is the total sequence numbers of one clone library).
0.3
0.2
0.1
0.0
0
100
200
300
400
500
Time (d)
Fig. 1 Nitrite consumption rates of cultures C1, C2, and C3 during the enrichment period. The enrichment period was divided into three phases according to the nitrite concentration of the influent. Symbols: C1 (black square), C2 (red circle), and C3 (blue triangle) (color figure online)
Appl Microbiol Biotechnol
and C3 (Fig. 2). The maximum n-damo specific activities of cultures C1, C2, and C3 were 0.8±0.1, 1.4±0.1, and 1.0± 0.1 μmol CH4 h−1 g−1 VSS, respectively. Culture C2 exhibited the highest n-damo specific activity among the three cultures, significantly higher than C1 (p value=0.0001) and C2 (p value=0.01). The ratios of the consumption rates of methane to nitrite for cultures C1, C2, and C3 were 3:8.9, 3:8.6, and 3:7.6, respectively, and the theoretical stoichiometric ratio is 3:8 in Eq. 1. Phylogenetic analysis of the 16S rRNA and pmoA genes Phylogenetic analysis of 16S rRNA genes indicated that only group A members were detected after 500 days of enrichment, whereas only group B members of the NC10 phylum were detected in the inocula (Fig. 3). This result further supported the hypothesis that the NC10 bacteria affiliated with group A perform n-damo process (Ettwig et al. 2009; Luesken et al. 2011b). The numbers of group A in the inocula might be below the detection limit, which led to only group B detected in all the three inocula. Only NC10 bacteria clustering in group A were enriched under the enrichment conditions provided by this study, whereas the NC10 bacteria clustering in group B were eliminated. All of the sequences obtained from the three enrichment cultures C1, C2, and C3 belonged to one cluster (differences between 0 and 0.9 %, 1 OTU) in group A. The sequences of cultures C1, C2, and C3 had identities of 96.7–97.2, 96.7–97.4, and 96.7–97.2 % to those of M. oxyfera, respectively. To detect the M. oxyfera-like bacteria on a functional level, pmoA genes were recovered from the inocula and enrichment 0.012
nitrite, methane (mmol)
0.011 0.010 0.009 0.008 0.007 0.006 0.005 0.004 0
-
C1_CH4
C1_NO2 -N
C2_CH4
C2_NO2 -N
C3_CH4
C3_NO2 -N
2
cultures achieved, since pmoA gene is often used as a functional marker for the detection of methanotrophs (McDonald et al. 2008). However, the pmoA gene of M. oxyfera is sufficiently different from that of aerobic methanotrophs, because it cannot be amplified by primer sets which commonly used to amplify the pmoA gene of the aerobic methanotrophs, in spite of a complete aerobic pathway to oxidize methane M. oxyfera has (Ettwig et al. 2010). Thus, a specific primer sets for the pmoA gene of M. oxyfera, cmo182F and cmo568R, was designed previously (Luesken et al. 2011c) and used to detect M. oxyfera-like bacteria here. Phylogenetic analysis of the pmoA genes of cultures C1, C2, and C3 revealed that all of the sequences obtained from the three cultures fell into in one cluster of NC10 bacteria (Fig. 4). This result was consistent with the observed phylogenetic relationship of the 16S rRNA genes of the NC10 bacteria in the enrichment cultures. With a cutoff of 7 % (Lüke and Frenzel 2011), the number of OTUs observed in cultures C1, C2, and C3 was 3, 3, and 4, respectively, and the cloning coverage was 100, 94.7, and 95.8 %, respectively. The sequences of cultures C1, C2, and C3 shared 96.7–97.2, 96.7– 97.4, and 96.7–97.2 % identity with the sequences of M. oxyfera, respectively. However, no pmoA gene sequences were detected from the inocula using the specific primer sets for M. oxyfera. Quantification analysis of NC10 bacteria The qPCR analysis of the inocula of cultures C1, C2, and C3 indicated that similar numbers of NC10 bacteria existed in the three inocula, and the numbers were 6.8±3.8×106, 9.4±1.9× 106, and 3.5±0.2×106 copies g−1 dry weight, respectively. After 500 days of enrichment, the numbers of NC10 bacteria in cultures C1, C2, and C3 reached 5.0±0.4×108, 6.1±0.1× 109, and 1.0±0.2×109 copies g−1 dry weight, respectively, which were 73, 646, and 290 times more than that of the inocula, respectively. The number of NC10 bacteria in culture C2 was significantly higher than those in cultures C1 and C3 (both p values
ENVIRONMENTAL BIOTECHNOLOGY
Effect of inoculum sources on the enrichment of nitrite-dependent anaerobic methane-oxidizing bacteria Zhanfei He & Chen Cai & Lidong Shen & Liping Lou & Ping Zheng & Xinhua Xu & Baolan Hu
Received: 6 July 2014 / Revised: 11 August 2014 / Accepted: 15 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Nitrite-dependent anaerobic methane oxidation (n-damo) is a newly discovered biological process that couples anaerobic oxidation of methane (AOM) to nitrite reduction. In this study, three different inocula, methanogenic sludge, paddy soil, and freshwater sediment were used to enrich n-damo bacteria in three sequencing batch reactors (SBRs), and three n-damo enrichment cultures, C1, C2 and C3, were obtained, respectively. After 500 days of incubation, Methylomirabilis oxyfera-like bacteria and n-damo activities were observed in cultures C1, C2, and C3, and the specific activities were 0.8±0.1, 1.4±0.1, and 1.0±0.1 μmol CH4 h−1 g−1 VSS, respectively. The copy numbers of 16S rRNA genes from cultures C1, C2, and C3 were 5.0±0.4×108, 6.1± 0.1×109, and 1.0±0.2×109 copies g−1 dry weight, respectively. The results indicated that paddy soil is an excellent inoculum for n-damo bacterial enrichment. This work expanded the alternative source of n-damo inoculum and benefited the further research of n-damo process. Keywords n-damo . Inoculum . Paddy soil . Methanogenic sludge . Freshwater sediment
Introduction Anaerobic oxidation of methane (AOM) mediated by microbes is crucial in its role as a sink of the global methane cycle, since it consumes approximately 7–25 % of the annual Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6033-8) contains supplementary material, which is available to authorized users. Z. He : C. Cai : L. Shen : L. Lou : P. Zheng : X. Xu : B. Hu (*) Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China e-mail: [email protected]
global methane production (Knittel and Boetius 2009). At present, investigations of AOM are mainly focused on AOM coupled to sulfate reduction (Knittel and Boetius 2009), but AOM coupled to nitrite reduction (Eq. 1), commonly named nitrite-dependent anaerobic methane oxidation (n-damo), also plays an important role in the methane cycle (Hu et al. 2014b). Raghoebarsing et al. (2006) successfully achieved an enrichment culture capable of AOM coupled to nitrite reduction from anoxic sediment from a canal, which was the first solid evidence for the existence of the n-damo process. The microorganisms responsible for n-damo process were affiliated with the uncultured NC10 phylum (Ettwig et al. 2009) and provisionally named Candidatus “Methylomirabilis oxyfera” (M. oxyfera) (Ettwig et al. 2010). 3CH4 þ 8NO2 − þ 8Hþ → 3CO2 þ 4N2 þ 10H2 O △G0’¼ −928 kJ mol−1 CH4
ð1Þ Hitherto, some n-damo cultures have been enriched successfully (Ettwig et al. 2009; Hu et al. 2009; Kampman et al. 2012, 2014; Luesken et al. 2011b; Raghoebarsing et al. 2006; Zhu et al. 2012), and the proportion of M. oxyfera-like bacteria ranged from 15 to 80 % of the total microorganisms in these cultures. The sources of inocula to enrich for n-damo cultures included freshwater sediment (Ettwig et al. 2009; Kampman et al. 2012; Raghoebarsing et al. 2006); wastewater sludge (Kampman et al. 2014; Luesken et al. 2011b); a mixture of freshwater lake sediment, anaerobic sludge, and return activated sludge (Hu et al. 2009); and minerotrophic peatland soil (Zhu et al. 2012). However, the source of n-damo inoculum was still limited; the effect of inoculum sources was unclear; and highly active n-damo culture was not achieved, which restricted the research of the n-damo process and its biotechnological application. In this study, methanogenic sludge, paddy soil, and freshwater sediment were used as inocula to enrich n-damo bacteria
Appl Microbiol Biotechnol
in three sequencing batch reactors (SBRs), and the obtained cultures were marked as C1, C2, and C3, respectively. Molecular techniques, including quantitative PCR (qPCR), 16S ribosomal RNA (rRNA) gene sequencing, pmoA gene sequencing, and fluorescence in situ hybridization (FISH), were employed to monitor changes in the microbial community before and after the cultivation period. The objective of this study was to determine the feasibility of using the three inocula above for the enrichment of n-damo bacteria, to expand the source of n-damo inocula, and to investigate the effect of inoculum source on the enrichment.
Materials and methods Inocula and medium Methanogenic sludge was sampled from a methanogenic granular sludge reactor; paddy soil was obtained from a paddy field with long-term fertilization (sampling depth 5–10 cm) in Zhejiang Academy of Agricultural Science (30° 18′ 41″ N, 120° 11′ 20″ E), Hangzhou, China; and freshwater sediment was obtained from Tiesha River (30° 15′ 59″ N, 120° 11′ 01″ E; sampling depth, top 20 cm of the sediment) located in the east of Hangzhou, China. The physicochemical characteristics of the three inocula are presented in Table S1. Natural freshwater was taken from the same location where the river sediment was sampled. This kind of natural freshwater with additive nitrite was used as the medium to enrich n-damo bacteria. During the 500 days of enrichment period, the influent concentration of nitrite raised from 0.5 to 1.5 mmol L−1; the influent pH was kept at 7.0–7.2; and the methane partial pressure in headspace was about 20 kPa.
Enrichment protocol Three identical SBRs were used to enrich n-damo bacteria (Fig. S1). The volume of each reactor was 2.5 L, consisted of 0.5 L of inoculum, 1.5 L of medium, and 0.5 L of headspace. Each reactor was mixed by a magnetic stirrer at 150 rpm and operated at 30±1 °C. The reactors were operated in sequencing batch mode with 72 h per cycle. Each cycle consisted of 0.5 h of medium supply, 70 h of incubation, 1 h of biomass settling, and 0.5 h of supernatant liquid discharge. During the medium supply period, pure Ar (99.999 %) was sparged into the reactors to maintain anaerobic condition. Then, 100 mL of methane (purity 99.99 %) was injected into the gas phase of each SBR. After settling, 1 L of supernatant was replaced by same volume of fresh medium.
Activity measurement The n-damo activities of cultures C1, C2, and C3 were measured by batch tests after 500 days of enrichment. Cultures sampled from the reactors were rinsed with nitrite-free medium three times, and then 10 mL of each settled culture was transferred into 72-mL serum bottles. Medium with 0.2 mmol L−1 of nitrite was added to reach a working volume of 50 mL. Subsequently, the serum bottles were flushed with pure Ar (99.999 %) for 15 min, sealed with stoppers, and added with 0.25 mL of pure methane (99.99 %). Assays were performed in a shaking table at 100 rpm and 30±0.5 °C. After 2 h of pre-incubation, 20 μL gas samples and 1 mL liquid samples were taken every 2 h from each bottle to analyze the concentrations of methane (in triplicate), nitrite, and nitrate during the next 10 h. After batch tests, volatile suspended solids (VSS) was measured to characterize the biomass of the cultures. DNA extraction and PCR amplification Samples (0.25 g) of each enrichment culture were collected. DNA extraction of each sample was performed as described previously (Hu et al. 2014a). The primers (Table S2) and amplification conditions used in this study were previously described by Luesken et al. (2011c). The PCR products were evaluated by electrophoresis in a 1.0 % agarose gel. Cloning and sequencing The PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Competent cells transformed with vectors were shaken in SOC medium for 1 h and then grown in LB medium containing ampicillin, X-Gal, and IPTG for 16 h at 37 °C. Clones with successful ligation were detected using the blue-white screening technique. Approximately 20 positive clones from each sample were sequenced by BGI-Shanghai (China). Phylogenetic analysis of 16S rRNA genes and pmoA genes Phylogenetic analysis of the obtained sequences was performed using Mega 4.0 (Tamura et al. 2007). The 16S rRNA and pmoA gene sequences were aligned with the ClustalW algorithm. Phylogenetic trees were constructed by the neighbor-joining method with p-distance correction, and the bootstrap values were based on 1,000 replicates. Operational taxonomic units (OTUs) for the determination of 16S rRNA and pmoA genes diversity were analyzed using 3 and 7 % differences in the nucleotide sequences (Lüke and Frenzel 2011), respectively, as determined by the DOTUR program (Schloss and Handelsman 2005). The
Appl Microbiol Biotechnol
The representative sequences reported in this study have been deposited in the GenBank database under accession numbers KC935359–KC935361 (16S rRNA) and KC935362– KC935371 (pmoA). Quantitative PCR analysis qPCR was performed with an iCycler iQ5 thermocycler and a real-time detection system (Bio-Rad, CA, USA) using specific primer set qP1F/qP1R (Ettwig et al. 2009). Thermal cycling program with annealing temperature of 63 °C were performed as previously described (Ettwig et al. 2009). The construction of the standard curve was conducted from a series of tenfold dilutions of plasmid DNA inserted by 16S rRNA genes of NC10 bacteria amplified through qPCR. Three replicates of each sample and plasmid DNA were conducted. The copy numbers of the samples were determined through a calculation based on comparison with the threshold cycle values of the standard curve. FISH
The enrichment period could be divided into three phases according to the nitrite concentration of the influent (Fig. 1). The nitrite concentration in the influent was 0.5, 1.0, and 1.5 mmol L−1 in phase I (0–180 days), phase II (181–400 days), and phase III (401–500 days), respectively. In phase I, the nitrite-reducing activity of culture C1 remained relatively constant, with an average value of 0.17 mmol L−1 day−1. In contrast, the nitrite-reducing activities of cultures C2 and C3 gradually decreased to about 0.05 and 0.09 mmol L−1 day−1, respectively. In phase II, the nitrite-reducing activity of culture C1 rapidly increased to 0.26 mmol L−1 day−1, mainly caused by the increase of influence nitrite that improved the biological activities. In contrast, during days 181–280, the nitritereducing activities of cultures C2 and C3 were at a low level (about 0.07 mmol L−1 day−1) but sharply increased to 0.20 and 0.23 mmol L−1 day−1, respectively, after 280 days. In phase III, the nitrite-reducing activities of cultures C1, C2, and C3 increased, and the average nitrite-reducing activities were 0.33, 0.32, and 0.34 mmol L−1 day−1, respectively. The doubling time of n-damo bacteria in the cultures were estimated based on the rapid increase in the nitrite-reducing activities using the method reported by He et al. (2013). The doubling times in cultures C1, C2, and C3 were estimated to be 3.2, 2.1, and 3.0 months from days 330–430, 330–430, and 250–430, respectively. n-damo activities of cultures The results of batch activity tests indicated that methane and nitrite could be simultaneously utilized by cultures C1, C2, 0.5
Phase I
Analytical methods The concentrations of ammonium, nitrite, and nitrate were measured using phenate method, colorimetric method, and ultraviolet spectrophotometric screening method (APHA 2005), respectively, and VSS levels were also determined according to the APHA standard methods (APHA 2005). Methane was quantified using an Agilent 6890 gas chromatograph (Agilent, USA) as described previously (Liebner and Wagner 2007). Transmission electron micrograph (TEM) was utilized as described in previous literatures (Jin et al. 2007, 2014). Statistical analysis was performed using IBM SPSS Statistics® Desktop version 19.0 (IBM, USA).
phase II
phase III
0.4
C1 C2 C3
-1
Samples of cultures C1, C2, and C3 were fixed and hybridized after 500 days of enrichment. Formamide (40 %) was combined with the following oligonucleotide probes: S-*DBACT-1027-a-A-18 (red), which is specific for dominant bacteria affiliated with the NC10 phylum (Raghoebarsing et al. 2006), and a mixture of EUB I-III (blue), which detects most bacteria (Daims et al. 1999). FISH images were obtained using a two-photon laser confocal microscope (Zeiss, LSM710 NLO, Germany).
Enrichment of n-damo bacteria
-1
Nucleotide sequence accession numbers
Results
nitrite consumption rate (mmol L d )
equation C=[1−(n1 /N)]×100 % was employed to determine the cloning coverage of each sample (n1 is the number of OTUs containing only one sequence; N is the total sequence numbers of one clone library).
0.3
0.2
0.1
0.0
0
100
200
300
400
500
Time (d)
Fig. 1 Nitrite consumption rates of cultures C1, C2, and C3 during the enrichment period. The enrichment period was divided into three phases according to the nitrite concentration of the influent. Symbols: C1 (black square), C2 (red circle), and C3 (blue triangle) (color figure online)
Appl Microbiol Biotechnol
and C3 (Fig. 2). The maximum n-damo specific activities of cultures C1, C2, and C3 were 0.8±0.1, 1.4±0.1, and 1.0± 0.1 μmol CH4 h−1 g−1 VSS, respectively. Culture C2 exhibited the highest n-damo specific activity among the three cultures, significantly higher than C1 (p value=0.0001) and C2 (p value=0.01). The ratios of the consumption rates of methane to nitrite for cultures C1, C2, and C3 were 3:8.9, 3:8.6, and 3:7.6, respectively, and the theoretical stoichiometric ratio is 3:8 in Eq. 1. Phylogenetic analysis of the 16S rRNA and pmoA genes Phylogenetic analysis of 16S rRNA genes indicated that only group A members were detected after 500 days of enrichment, whereas only group B members of the NC10 phylum were detected in the inocula (Fig. 3). This result further supported the hypothesis that the NC10 bacteria affiliated with group A perform n-damo process (Ettwig et al. 2009; Luesken et al. 2011b). The numbers of group A in the inocula might be below the detection limit, which led to only group B detected in all the three inocula. Only NC10 bacteria clustering in group A were enriched under the enrichment conditions provided by this study, whereas the NC10 bacteria clustering in group B were eliminated. All of the sequences obtained from the three enrichment cultures C1, C2, and C3 belonged to one cluster (differences between 0 and 0.9 %, 1 OTU) in group A. The sequences of cultures C1, C2, and C3 had identities of 96.7–97.2, 96.7–97.4, and 96.7–97.2 % to those of M. oxyfera, respectively. To detect the M. oxyfera-like bacteria on a functional level, pmoA genes were recovered from the inocula and enrichment 0.012
nitrite, methane (mmol)
0.011 0.010 0.009 0.008 0.007 0.006 0.005 0.004 0
-
C1_CH4
C1_NO2 -N
C2_CH4
C2_NO2 -N
C3_CH4
C3_NO2 -N
2
cultures achieved, since pmoA gene is often used as a functional marker for the detection of methanotrophs (McDonald et al. 2008). However, the pmoA gene of M. oxyfera is sufficiently different from that of aerobic methanotrophs, because it cannot be amplified by primer sets which commonly used to amplify the pmoA gene of the aerobic methanotrophs, in spite of a complete aerobic pathway to oxidize methane M. oxyfera has (Ettwig et al. 2010). Thus, a specific primer sets for the pmoA gene of M. oxyfera, cmo182F and cmo568R, was designed previously (Luesken et al. 2011c) and used to detect M. oxyfera-like bacteria here. Phylogenetic analysis of the pmoA genes of cultures C1, C2, and C3 revealed that all of the sequences obtained from the three cultures fell into in one cluster of NC10 bacteria (Fig. 4). This result was consistent with the observed phylogenetic relationship of the 16S rRNA genes of the NC10 bacteria in the enrichment cultures. With a cutoff of 7 % (Lüke and Frenzel 2011), the number of OTUs observed in cultures C1, C2, and C3 was 3, 3, and 4, respectively, and the cloning coverage was 100, 94.7, and 95.8 %, respectively. The sequences of cultures C1, C2, and C3 shared 96.7–97.2, 96.7– 97.4, and 96.7–97.2 % identity with the sequences of M. oxyfera, respectively. However, no pmoA gene sequences were detected from the inocula using the specific primer sets for M. oxyfera. Quantification analysis of NC10 bacteria The qPCR analysis of the inocula of cultures C1, C2, and C3 indicated that similar numbers of NC10 bacteria existed in the three inocula, and the numbers were 6.8±3.8×106, 9.4±1.9× 106, and 3.5±0.2×106 copies g−1 dry weight, respectively. After 500 days of enrichment, the numbers of NC10 bacteria in cultures C1, C2, and C3 reached 5.0±0.4×108, 6.1±0.1× 109, and 1.0±0.2×109 copies g−1 dry weight, respectively, which were 73, 646, and 290 times more than that of the inocula, respectively. The number of NC10 bacteria in culture C2 was significantly higher than those in cultures C1 and C3 (both p values
