Optimum O2:CH4 Ratio Promotes the Synergy between Aerobic Methanotrophs and Denitrifiers to Enhance Nitrogen Removal

The O₂:CH₄ ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O₂:CH₄ ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O₂:CH₄ ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems.

Introduction

Biological denitrification, following nitrification, is widely used to remove nitrogen from wastewater. It includes four reduction steps: (1) from nitrate (${\text{NO}}_3^{-}$) to nitrite (${\text{NO}}_2^{-}$), (2) from ${\text{NO}}_2^{-}$ to nitric oxide (NO), (3) from NO to nitrous oxide (N₂O) and (4) from N₂O to dinitrogen (N₂) (Zumft, 1997). Theoretically, ${\text{NO}}_3^{-}$ and ${\text{NO}}_2^{-}$ can be completely reduced to N₂ in the presence of enough carbon sources if the microbes are equipped with full set of denitrification genes (Rittmann and McCarty, 2001). Among the four steps of denitrification, the reduction of soluble nitrite by nitrite reductase into gas is the key step and considered as the symbol of permanent removal of nitrogen from the aquatic ecosystem (Saunders and Kalff, 2001; Philippot and Hallin, 2005). Currently, carbons in the forms of methanol, ethanol and acetate are frequently supplemented for complete denitrification in wastewater treatment systems. However, the addition of external carbon sources inevitably increases the operational cost of wastewater treatment. Methane (CH₄), a greenhouse gas, is readily available in many wastewater treatment plants and landfills and has a potential as an electron donor to replace traditional carbon sources for denitrification in nitrate-contaminated wastewater treatment (Modin et al., 2007). It was successfully demonstrated as a carbon source for denitrification in the presence of oxygen for the first time in 1978 (Rhee and Fuhs, 1978). This process was defined as aerobic methane oxidation coupled with denitrification (AME-D) (all the following discussions about this process are based on wastewater treatment systems). The AME-D process is a promising and realistic alternative to conventional biological treatment of nitrate-rich wastewaters. However, the nitrogen removal performance of AME-D systems is required to be higher than conventional systems for practical applications. Basic knowledge regarding operational parameters that affect the AME-D process is highly needed for the system process design.

The AME-D process is a synergistic collaboration between aerobic methanotrophs and denitrifying bacteria. Aerobic methanotrophs are a group of microorganisms capable of utilizing CH₄ as a carbon and energy source. These microbes can oxidize CH₄ to carbon dioxide (CO₂) in the presence of oxygen (O₂). Metabolic pathways of methane oxidation in aerobic methanotrophs are comprehensively summarized by other researchers (Trotsenko and Murrell, 2008; Zhu et al., 2016; Figure 1). Briefly, CH₄ is initially catalyzed by methane monooxygenase (MMO), soluable MMO (sMMO) or particulate MMO (pMMO), to produce methanol as the first intermediate. Afterwards, methanol is transformed into formaldehyde by methanol dehydrogenase. Formaldehyde may be assimilated into biomass through the ribulose monophosphate pathway or the serine pathway, releasing multi-carbon intermediates such as acetate and citrate. Alternatively, formaldehyde can be dissimilated to CO₂ via formate for energy production. It was demonstrated that dissolved organic intermediates, such as methanol (Meschner and Hamer, 1985), formaldehyde (Liu et al., 2014), acetate (Costa et al., 2000), which were released during aerobic methane oxidation, could be used as carbon sources for co-existing denitrifiers. On the metabolic pathways, denitrification in the AME-D process consists of two critical steps that may occur simultaneously or sequentially. The first is methane oxidation to release carbon sources, and the second is the use of these carbon compounds for denitrification (i.e., nitrate or nitrite removal). Therefore, operational parameters that affect the organic carbons excreted by methanotrophs for denitrification would have a critical impact on the nitrogen removal of AME-D process. Because of the well-known inhibition of excessive O₂ on denitrification (Zumft, 1997), supply of enough electron donors and well-control of O₂ level are two critical strategies to improve nitrogen removal and promote complete denitrification (${\text{NO}}_3^{-}$-${\text{NO}}_2^{-}$-NO-N₂O-N₂) in the AME-D systems.

FIGURE 1

Figure 1. Traditional metabolism of methane in aerobic methanotrophs (modified from Zhu et al., 2016). pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase; MDH, methanol dehydrogenase; FDH^a, formaldehyde dehydrogenase; FDH^b, formate dehydrogenase; RuMP, ribulose monophosphate pathway. PQQ, pyrroloquinolinequinone; NAD⁺, oxidized form of nicotinamide-adenine dinucleotide; NADH, reduced form of nicotinamide-adenine dinucleotide.

It has been demonstrated that the O₂:CH₄ ratio is an essential parameter to regulate the carbon flow from CH₄ to biomass and CO₂, which impacts the production of carbon sources for nitrogen removal in the AME-D process. Morinaga et al. (1979) and Costa et al. (2000) discovered that methanotrophic strains could excrete formaldehyde and acetate, when the O₂:CH₄ ratio was lower than 1.0 (i.e., oxygen-limited). In contrast, no organic metabolites were detected when O₂:CH₄ ratio was higher than 1.0 (i.e., methane-limited). Kalyuzhnaya et al. (2013) further studied the pathway of intermediates production by aerobic methanotrophs under oxygen-limited conditions and discovered that acetate, lactate, and even hydrogen could be released during novel fermentation-related methanotrophy. Besides, oxygen is another critical factor to influence nitrogen removal in the AME-D process. Aerobic methanotrophs require enough O₂ to utilize CH₄, whereas excessive O₂ will inhibit denitrifiers to reduce nitrogen (Modin et al., 2007). To date, environmental conditions that are favorable for both aerobic methanotrophs and denitrifiers are still unclear. Determination of an optimal O₂:CH₄ ratio which balances the requirements of carbon source and O₂ could be an effective way for improving denitrifying performance in the AME-D process.

The aim of this research was to investigate the impact of the O₂:CH₄ ratio on nitrogen removal in the AME-D process and to determine the optimal ratio resulting in the highest nitrogen removal performance. It was hypothesized that intermediates released by methanotrophs primarily control nitrogen removal when O₂:CH₄ ratio is below the optimal value, whereas O₂ level mainly controls nitrogen removal when O₂:CH₄ ratio is over the optimal. According to the stoichiometric (Equations 1 and 2) of the AME-D process (Zhu et al., 2016), the O₂:CH₄ ratio should be maintained below 1.1:1 (more specifically 0, 0.05, 0.25, and 1.0 in this study) to avoid excessive O₂ inhibition on denitrification.

$\begin{array}{l}{\text{CH}}_4+1.1{\text{O}}_2+0.7{\text{2NO}}_3^{-}+0.72{\text{H}}^{+}=0.36{\text{N}}_2+{\text{CO}}_2\\ +2.36{\text{H}}_2\text{O}& (1)\end{array}$

$\begin{array}{l}{\text{CH}}_4+1.1{\text{O}}_2+1.2{\text{NO}}_2^{-}+1.2{\text{H}}^{\text{+}}=0.6{\text{N}}_2+{\text{CO}}_2\\ +2.6{\text{H}}_2\text{O}& (2)\end{array}$

Materials and Methods

Sludge Preparation

The sludge used in this study was collected from an AME-D culture that was enriched for more than 1 year in a batch bioreactor with continuous CH₄ supply. The sludge was pre-incubated in a nitrite-containing medium under anoxic conditions in the dark at 25°C for 3 days to eliminate residual organic carbon sources. Then the sludge was centrifuged at 3,300 g for 5 min and the supernatant was discarded. The sludge pellet was resuspended and washed in sterile phosphate buffer solution (pH = 6.8) for three times to further remove extracellular organic carbons. Subsequently, it was centrifuged at higher speed of 14,000 g for 10 min to ensure complete precipitation of suspended cells. The experiments were initiated when no organic carbon was present in the supernatant. The organic carbon was determined with a Total Organic Carbon (TOC) analyzer (MultiN/C3100, Analytikjena, Germany). After all residual organics were removed, the sludge was re-suspended in the basal medium (Liu et al., 2014) at a mixed liquor suspended solid (MLSS) concentration of 17,735 mg/L. The basal medium contained the following components in 1 liter (L) distilled water: 1,250 mg KHCO₃, 50 mg KH₂PO₄, 300 mg CaCl₂·2H₂O, 200 mg MgSO₄·7H₂O, 345 mg NaNO₂, 1.0 mL acidic trace element solution and 1.0 mL alkaline trace element solution. Constituents of the acidic and alkaline trace element solution were described by Liu et al. (2014). Inorganic nitrogenous compounds were prepared from NaNO₂ to result in a concentration of ${\text{NO}}_2^{-}$-N in the basal medium of 70 mg/L. Ammonium (${\text{NH}}_4^{+}$) and ${\text{NO}}_3^{-}$ were also supplied at low concentrations, 0.07 and 3.82 mg/L, respectively, to serve as nitrogen sources for microbes in the AME-D system. The pH of the medium was 7.2.

Batch Experiment

Batch experiment was conducted using 10 mL of the prepared sludge and 20 mL of the basal medium in a 150 mL glass vial. Freshly prepared mixture of the basal medium and the sludge was sparged with CH₄ (99.99%) for 5 min. The vial was crimp-sealed with a butyl rubber stopper. Different volumes of CH₄ (0, 6, 24, and 60 mL) were withdrawn from the headspace of the vials with a gas-lock syringe. Afterwards, the same volume of pure O₂ (99.99%) was injected into the headspace of vials. The final compositions of O₂ and CH₄ in the gas mixtures of each treatment were shown in Table 1. Un-inoculated media was used as blank controls to test for leakage and non-biological chemical transformations. Triplicate samples were incubated at 28°C in shaking incubator at 180 rpm in the dark for 60 h. CH₄ and nitrogen (${\text{NO}}_3^{-}$-N, ${\text{NO}}_2^{-}$-N and ${\text{NH}}_4^{+}$-N) consumptions and intermediate production were monitored in each treatment.

TABLE 1

Table 1. Experimental set up for aerobic methane oxidation coupled with denitrification (AME-D) process.

Chemical Analysis

The ${\text{NO}}_3^{-}$-N, ${\text{NO}}_2^{-}$-N, and ${\text{NH}}_4^{+}$-N concentrations were determined by ultraviolet spectrophotometry (UV-5300PC, METASH, China). The measurement of volatile suspended solid (VSS) was performed according to standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation, 1989). In this study, nitrite removal expressed as mmol nitrite consumed per gram of biomass per day i.e., mmol ${\text{NO}}_2^{-}$-N/gVSS/d. To quantify CH₄ content in the headspace of each vial, a sample of 0.5 mL was removed from the headspace of the vial with a gas-lock syringe. The sample was analyzed with a gas chromatograph (GC) equipped with a thermal conductivity detector under the conditions previously described by Zhang et al. (2014). Methane oxidation activity was expressed as CH₄ consumed per gram of biomass per day i.e., mmol CH₄/gVSS/d (Wang et al., 2008). NO concentration was measured by GC-mass spectrometry (6890N GC-5973MS, Agilent, USA) with the methods described by Leone et al. (1994). N₂O concentration in the vial headspace was determined by a GC (GC-14B, Shimadzu, Japan) equipped with an electron capture detector and a Porapak Q column maintained at 330 K. Intermediate (methanol, formaldehyde, formate, acetate, and other potential organics) concentrations were determined with a high performance liquid chromatograph using the methods described by Thalasso et al. (1997).

DNA Extraction

Sludge samples were analyzed for DNA before and after incubation. Three independent DNA extractions of each treatment were performed from 30 mg of sludge using a FastDNA SPIN Kit for Soil (MP Biomedical, LLC, Ohio, USA). The extraction was performed according to the manufacturer's instructions. The concentrations and the quality of DNA samples were measured with a Nanodrop analyzer (Thermo Scientific, Wilmington, DE, USA). Extracted DNA was stored at −20°C prior to subsequent analyses.

Quantification of Functional Genes

The abundance of aerobic methanotrophs and denitrifiers was estimated through quantitative PCR (q-PCR). Due to the low level of mmoX-harboring methanotrophs in the enrichment and high enough Cu concentration (2 μM) in the medium to inhibit the expression of mmoX gene (Takeguchi et al., 1997), the function of sMMO encoded by mmoX gene was considered to be negligible. Therefore, only pmoA gene encoding a subunit of pMMO was used to investigate the abundance variation of aerobic methanotrophs in this study. For denitrifiers, the nirK gene encoding copper nitrite reductase and the nirS gene encoding cytochrome cd₁-containing nitrite reductase were used as the biomarkers. The quantification was based on the intensity of SYBR Green dye fluorescence, which can bind to double-stranded DNA. Standard curve for each gene were generated using a 10-fold dilution series of the linearized plasmid standard (10⁻¹–10⁻⁶ ng) ranging from 10⁸ to 10³ copies. Each qPCR assay (25 μL) included 12.5 μL of 2 × SYBR Premix Ex Taq (Takara, Dalian, China), 1 μL of each forward and reverse primer (20 μM), either 1 μL of template DNA or the standard vector plasmid of the clones grown as single cellular suspension. The optimized thermal conditions and primers used for each gene can be viewed in the Supplementary Table 1. All real-time PCR assays were performed in triplicate for each sample in a Bio-Rad CFX1000 Thermal Cycler. All PCR runs included negative controls that did not contain DNA templates. The gene copy numbers were determined by comparing threshold cycles obtained in each PCR run with those of known standard DNA concentrations. Standard curves were obtained using serial dilutions of linearized plasmids containing cloned pmoA, nirK, and nirS genes.

Statistical Analysis

All data are presented as means and standard deviations. Analysis of variance and least significant difference (LSD) tests at the 5% level were used to determine the statistical significance of different treatments. Any differences with p ≥ 0.05 were not considered as statistically significant. The relationships between nirK/nirS gene copies and the nitrite removals at four treatments were tested with linear regression analyses using SPSS 20.0 for Windows (SPSS Inc., Chicago, IL).

Results

Methane Oxidation Activity and Intermediates Accumulation at Different O₂:CH₄ Ratios

Methane oxidation activity and concentrations of extracellular metabolites (methanol, formaldehyde, formate, and acetate, etc.) were determined under four different O₂:CH₄ ratios. As shown in Figure 2, methane oxidation activity substantially decreased from 277.80 mmol/gVSS/d to 21.06 mmol/gVSS/d when the O₂:CH₄ ratio was increased from 0 to 1 (p < 0.05). With the exception of treatment 1 at an O₂:CH₄ ratio of 0, a similar trend was observed for qPCR data of the pmoA gene. The pmoA gene abundance decreased almost an order of magnitude (from 9.36 × 10⁹ to 1.63 × 10⁹ copies per g dry biomass) when the O₂:CH₄ ratio was increased from 0.05 to 1 (Figure 2). The three primary metabolites observed in the bulk media were formaldehyde, acetate and citrate. Their concentrations went up from 42 to 76 μg/L (formaldehyde), 11 to 45 μg/L (acetate) and 0 to 28 μg/L (citrate), respectively, when O₂:CH₄ ratio was increased from 0 to 1 (Figure 3). Methanol, formate and butyrate were considered as three trace metabolites and their concentrations were lower than 1.20 μg/L (Supplementary Figure 1). However, methanol were not detected in the treatment 1 in the absence of O₂.

FIGURE 2

Figure 2. Methane oxidation activities of the consortia and the abundance of pmoA gene at different O₂:CH₄ ratios in aerobic methane oxidation coupled with denitrification (AME-D) process. T1, Treatment 1 (0% O₂ and 100% CH₄); T2, Treatment 2 (5% O₂ and 95% CH₄); T3, Treatment 3 (20% O₂ and 80% CH₄); T4, Treatment 4 (50% O₂ and 50% CH₄).

FIGURE 3

Figure 3. Accumulation of acetate, citrate and formaldehyde in the liquid at different O₂:CH₄ ratios in aerobic methane oxidation coupled with denitrification (AME-D) process. T1, Treatment 1 (0% O₂ and 100% CH₄); T2, Treatment 2 (5% O₂ and 95% CH₄); T3, Treatment 3 (20% O₂ and 80% CH₄); T4, Treatment 4 (50% O₂ and 50% CH₄).

Nitrite Removal at Different O₂:CH₄ Ratios

The concentrations of ${\text{NO}}_2^{-}$-N, ${\text{NH}}_4^{+}$-N and ${\text{NO}}_3^{-}$-N were measured at the beginning and the end of incubation. Results showed that concentrations of ${\text{NH}}_4^{+}$-N and ${\text{NO}}_3^{-}$-N decreased slightly by the end of the experiment and were detected in all final samples (Supplementary Table 2). There was a jump in the nitrite removal from 0.53 mmol ${\text{NO}}_2^{-}$-N/gVSS/d to 7.32 mmol ${\text{NO}}_2^{-}$-N/gVSS/d when the O₂:CH₄ ratio was increased from 0 to 0.25 (Figure 4). However, the nitrite removal decreased by 53.8% as the O₂:CH₄ ratio was increased from 0.25 to 1 (p < 0.05). The amount of reduced nitrogen released as NO was very low (0.13–0.32 μM; Table 2). In addition, the percentage of the reduced nitrogen emitted as N₂O decreased from 37.96 to 12.30% when the O₂:CH₄ ratio increased from 0 to 0.25 (Table 2). This percentage at the O₂:CH₄ ratio of 1 was about 2 times higher than that at the O₂:CH₄ ratio of 0.25, while this difference was insignificant (p > 0.05; Table 2). The highest nitrite removal and the low percentage of NO-N and N₂O-N in total reduced nitrogen were observed at the O₂:CH₄ ratio of 0.25. Based on the results, the O₂:CH₄ ratio of 0.25 was proposed as the optimal ratio for this AME-D system.

FIGURE 4

Figure 4. Nitrite removal of the consortia and the abundance of functional genes at different O₂:CH₄ ratios in aerobic methane oxidation coupled with denitrification (AME-D) process. T1, Treatment 1 (0% O₂ and 100% CH₄); T2, Treatment 2 (5% O₂ and 95% CH₄); T3, Treatment 3 (20% O₂ and 80% CH₄); T4, Treatment 4 (50% O₂ and 50% CH₄).

TABLE 2

Table 2. Reduction of ${\text{NO}}_2^{-}$-N and accumulation of NO-N and N₂O-N at different O₂:CH₄ ratios in aerobic methane oxidation coupled with denitrification (AME-D) process.

Nitrite reduction is catalyzed by nitrite reductase which are found in two different forms: copper nitrite reductase encoded by nirK gene and cytochrome cd₁-containing nitrite reductase encoded by nirS gene (Wang et al., 2014). Investigating the difference in nirK and nirS gene abundance might provide further evidences for the variation of nitrite removal. The abundance of nirK gene was more than doubled as the O₂:CH₄ ratio was increased from 0 to 0.25 (from 3.47 × 10¹⁰ copies/gVSS up to 7.91 × 10¹⁰ copies/gVSS). However, it descended to 6.23 × 10¹⁰ copies/gVSS as the O₂:CH₄ ratio was further raised to 1. In contrast, copy numbers of nirS gene displayed no significant change (a slight increase from 1.82 copies/gVSS to 2.34 × 10¹¹ copies/gVSS) when O₂:CH₄ ratio was increased from 0 to 0.25. However, it went down significantly to 7.10 × 10¹⁰ copies/gVSS at higher O₂:CH₄ ratio of 1. In addition, the linear correlation analysis revealed that nirK gene copies were positively correlated with nitrite removal (r² = 0.8639, p = 0.0707), whereas nirS gene copies had a slightly negative correlation with the nitrite removal (r² = 0.0121, p = 0.8899; Figure 5). If the nitrite removal was considered as the representation of the denitrifying conditions in the corresponding treatment, the higher nitrite removal indicated the better denitrifying conditions in this treatment. Thereby, the linear coorelation analyses suggest that nirK-type denitrifiers might be more responsive to the denitrifying conditions than nirS-type denitrifiers (Yoshida et al., 2010).

FIGURE 5

Figure 5. The relationships between nitrite removal and the abundance of nirK/nirS genes in aerobic methane oxidation coupled with denitrification (AME-D) process. Linear regressions were used to test the correlation between nitrite removal and the abundance of nirK/nirS genes.

Discussion

The Optimal O₂:CH₄ Ratio with Highest Nitrite Removal of AME-D Process

The results from the batch experiment showed that the peak nitrite removal (7.32 mmol ${\text{NO}}_2^{-}$-N/gVSS/d) was obtained at an O₂:CH₄ ratio of 0.25. As ${\text{NH}}_4^{+}$-N and ${\text{NO}}_3^{-}$-N are the preferred microbial nitrogen sources (Modin et al., 2007), their presence throughout the incubation period (Supplementary Table 2) indicated that nitrite was negligibly consumed as a nitrogen source for assimilation. Therefore, nitrite removal from the media was through dissimilatory, i.e., denitrification. Additionally, the low percentage of the consumed ${\text{NO}}_2^{-}$-N emitted as N₂O-N (12.30%) and NO-N (0.20%, Table 2) at the O₂:CH₄ ratio of 0.25 indicated that enough carbon sources provided by the methanotrophs may allow denitrifiers to reduce almost 87.5% of ${\text{NO}}_2^{-}$-N through complete denitrification. At a lower O₂:CH₄ ratio of 0.25, it was consistent with our hypothesis that nitrite removal were not inhibited by O₂, and were stimulated by the increased carbon provided by methanotrophic metabolism. However, nitrite removal was less effective at higher O₂:CH₄ ratios with higher oxygen concentrations, which was corroborated by the lower abundance of denitrifying genes (Figure 4).

The conclusion that nitrite removal was stimulated by carbon sources released from aerobic methane oxidation at a O₂:CH₄ ratio lower than or equal to 0.25 could be supported by the variation of intermediate concentrations among four treatments. Concentrations of accumulated intermediates were higher and higher in the bulk media with the O₂:CH₄ ratios increasing from 0 to 0.25 (Figure 3). It may be attributed to the increased O₂ concentration in these treatments. Morinaga et al. (1979) and Costa et al. (2001) demonstrated that the O₂ level would significantly impact the consumption and production of metabolites in methane metabolism. However, the effect was not unambiguously determined. Morinaga et al. (1979) observed formaldehyde accumulation under oxygen-limited conditions, whereas Costa et al. (2001) discovered that formaldehyde accumulated under oxygen-excessive conditions. Our results indicated that the increased oxygen level promoted intermediates accumulation in methane metabolism. Low level of available carbon sources for denitrifiers in Treatment 1 resulted in the lowest nitrite removal. Once O₂ was largely induced in the headspace in Treatment 2 and 3, concentrations of intermediates increased in the liquid bulk. The observation of simultaneous higher nitrite removal and lower ratio of accumulated N₂O-N:consumed ${\text{NO}}_2^{-}$-N indicated that complete denitrifying activity was improved by these accumulated intermediates.

The ability of denitrifiers to resist the inhibition of O₂ at the optimal O₂:CH₄ ratio may be due to two reasons. Firstly, the abundance of nirK-harboring denitrifiers was the highest among the four treatments and these denitrifiers are able to tolerate higher O₂ levels (Desnues et al., 2007). Secondly, the sludge aggregation/granulation in anoxic micro-environments can lessen the O₂ exposure of the denitrifiers. As shown in Figure 6, at the O₂:CH₄ ratio of 0.25, suspended biomass formed granule-like agglomerates with an average diameter of about 2 mm, while such effect did not occur for the O₂:CH₄ ratios of 0, 0.05, and 1. Sludge aggregation has also been observed in an AME-D system with an optimized O₂ level that had the highest nitrogen removal rate (Thalasso et al., 1997). It can be therefore concluded that sludge aggregation at an optimal O₂:CH₄ ratio could improve nitrite removal. The spatial distribution of microorganisms and O₂ level within the aggregates should be investigated in the future.

FIGURE 6

Figure 6. Configuration of activated sludge in four treatments at the end of incubation during aerobic methane oxidation coupled with denitrification (AME-D) process. The phenomenon of aggregation is apparent in treatment 3 (T3) while activated sludge in other treatments were dispersive. T1, Treatment 1 (0% O₂ and 100% CH₄); T2, Treatment 2 (5% O₂ and 95% CH₄); T3, Treatment 3 (20% O₂ and 80% CH₄); T4, Treatment 4 (50% O₂ and 50% CH₄).

With regard to the effect of the O₂:CH₄ ratio on denitrification, Sun et al. (2013) observed a similar phenomenon that the O₂:CH₄ ratio affected nitrogen removal of AME-D process in a membrane biofilm bioreactor (MBfR). Their optimal O₂:CH₄ ratio of 1.5 was considerably higher than the one in this study. It is likely that the spatial arrangement of the microbial community within a well-developed biofilm would allow for greater tolerance to O₂, as compared to a suspended culture. They speculated that greater metabolite excretion by the methanotrophs improved nitrate removal performance initially, and that excessive O₂ caused the significant drop in the denitrifying rate when O₂:CH₄ ratio increased to 2.0. The results from this study corroborate the trend that Sun et al. (2013) observed.

Methane oxidation activity dramatically decreased when O₂:CH₄ ratio increased from 0 to 1 (Figure 2). It was probably due to the large change in methane availability. Li et al. (2014) observed a similar trend with methanotrophic activity of landfill cover soils. The methane oxidation activity at O₂:CH₄ ratio of 0.25 (4 × 10⁴ ppmv O₂/2 × 10⁵ ppmv CH₄) was almost 4-fold higher than that at O₂:CH₄ ratio of 4.00 (2 × 10⁵ ppmv O₂/5 × 10⁴ ppmv CH₄) (Supplementary Table 3). In their research, methane oxidation activity was much more sensitive to CH₄ than O₂, and a drop of CH₄ concentration would result in a simultaneous decrease of methane oxidation activity. Additionally, copy numbers of the pmoA gene decreased as the methane concentration decreased (Baani and Liesack, 2008; Li et al., 2014). In the current research, CH₄ concentration declined from 100 to 50% and O₂ concentrations rose from 0 to 50% for the ratios tested. This suggests that a decrease in methane oxidation activity with a substantial increase in O₂:CH₄ ratio is plausible. However, it is unexpected that the high pmoA gene copy numbers and methane oxidation activity were observed in Treatment 1 in the absence of O₂. Rechecking the gaseous composition of the headspace in this treatment at the beginning of the incubation, it was discovered that trace amount (0.012%) of O₂ can still be detected after the sludge was sparged with pure CH₄ (99.99%). This trace level of O₂ may result in the observed CH₄ consumption. However, further investigations are still required to focus on examining if anaerobic methane oxidation contributed to CH₄ consumption when this trace amount of O₂ was depleted.

Thermodynamic Speculation for Metabolic Pathways of AME-D Process

Acetate, citrate, and formaldehyde were detected as three primary compounds detected during the AME-D process, while methanol, formate, and butyrate occurred in trace quantities. It is difficult to postulate which were the main substrates for the denitrifiers as their levels of consumption relative to production were not known. However, thermodynamic analysis of AME-D process may provide useful information for speculating the actual metabolic pathway (all of the following thermodynamic analyses are based on aerobic methane oxidation coupled with complete denitrification).

Methanol is considered as the most effective intermediate for denitrification according to the review of the AME-D process (Zhu et al., 2016). The reactions (Equations 3–5) related to energy production contained in AME-D process using ${\text{NO}}_2^{-}$ as the denitrifying electron acceptor were shown in Table 3. These equations are based on one electron equivalent (eeq). Assuming that at least X (<1) mol of methanol is needed by aerobic methanotrophs for their requirement of cell synthesis and maintenance when one mole of CH₄ is oxidized to methanol, the remaining part of methanol can be used for denitrification as the electron donor. An energy-balanced equation for the AME-D process can be described as Equation (6).

TABLE 3

Table 3. Stoichiometric equations in aerobic methane oxidation coupled with denitrification (AME-D) process based on theoretical hypotheses.

$\begin{array}{cc}6·\Delta G_{Eq.\left(4\right)}·\epsilon ·X+2·\left[\Delta G_{Eq.\left(3\right)}-0.5·\Delta G_{xy}\right]+\Delta G_m=0& (6)\end{array}$

Where ε, is the energy transfer efficiency a value of 0.37 (McCarty, 2007). [ΔG_Eq.(3) – 0.5ΔG_xy] denotes the net energy production for the oxidation from CH₄ to methanol after considering the energy input for the mono-oxygenase and the required reducing equivalent, which equals to 47.26 kJ/eeq. ΔG_m represents the minimum maintenance energy requirement with a value of about 8.1 kJ/mol oxidized CH₄ (Modin et al., 2007). The exhaustive description of calculation processes for [ΔG_Eq.(3) – 0.5ΔG_xy] and ΔG_m is presented by Zhu et al. (2016). From Equation (6), the value derived for X is 0.40. Because the energy required by cell synthesis has not been considered during the above calculation, the theoretical maximum quantity of methanol used by denitrifiers was 0.60 mol. This means that the maximum proportion of methanol that can be captured by denitrifiers is 60%. The overall reaction of AME-D process in the presence of ${\text{NO}}_2^{-}$ was described as Equation (7), a combination of the proposed value and Equations (3–5).

$\begin{array}{l}{\text{CH}}_4+\frac{11}{10}{\text{O}}_2+\frac{6}{5}{\text{NO}}_2^{-}+\frac{6}{5}{\text{H}}^{+}=\frac{3}{5}{\text{N}}_2+{\text{CO}}_2+\frac{13}{5}{\text{H}}_2\text{O}\\ \Delta {\text{G}}^{0^{\prime }}=-867.86\text{kJ}/\text{mol}{\text{CH}}_4& (7)\end{array}$

All six substrates detected in this study were potential carbon sources for denitrifiers. In order to understand which substrates were likely the functional intermediates, thermodynamic analysis was performed based on the chemical data associated with the O₂:CH₄ ratio of 0.25. During the process of thermodynamic derivation, all of organics detected in the bulk liquid were individually chosen as the trophic link of aerobic methane oxidation and denitrification. After several iterations, the same general equation (Equation 8) for the AME-D process, which was in agreement with CH₄ and ${\text{NO}}_2^{-}$ consumption was obtained through three different approaches:

(1) 4.34% of consumed CH₄-C flowed to denitrifiers using methanol as the trophic link;

(2) 5.22% of consumed CH₄-C flowed to denitrifiers using butyrate as the trophic link;

(3) 6.52% of consumed CH₄-C flowed to denitrifiers using formaldehyde as the trophic link. This means that methanol, butyrate and formaldehyde released by aerobic methanotrophs were three possible intermediates that could be used as carbon sources by denitrifiers at an O₂:CH₄ ratio of 0.25. However, acetate could not be an active carbon source under this condition in our study, although it was a feasible electron donor with highest denitrifying potential (Hallin and Pell, 1998). This conclusion is further supported by the increased copy numbers of nirK genes, which would have decreased if acetate was the main active electron donor for denitrifiers (Li et al., 2015).

$\begin{array}{l}{\text{CH}}_4+\frac{89}{46}{\text{O}}_2+\frac{2}{23}{\text{NO}}_2^{-}+\frac{2}{23}{\text{H}}^{+}=\frac{1}{23}{\text{N}}_2+{\text{CO}}_2+\frac{47}{23}{\text{H}}_2\text{O}\\ \Delta {\text{G}}^{0^{\prime }}=-821.65\text{kJ}/\text{mol}{\text{CH}}_4& (8)\end{array}$

Based on the above analysis, it was evident that the percentage of carbon flow from methane to denitrification (4.34–6.52%) was much lower than the ideal flow (60%). Further improvement in the carbon flow is vital to enhance the AME-D denitrification rates.

Implication of O₂:CH₄ Ratio Control for Nitrogen Removal in AME-D Process

The effect of the O₂:CH₄ ratio was demonstrated to significantly impact nitrogen removal during the AME-D process through contribution of the carbon metabolites generated by the methanotrophs and oxygen inhibition. To date, most studies have considered only the individual impact of O₂ on the apparent nitrogen removal rate of the AME-D process (Werner and Kayser, 1991; Thalasso et al., 1997; Modin et al., 2010), whereas the combined effect of O₂ and CH₄ and associated mechanisms are rarely investigated. It is necessary to address the impact of O₂:CH₄ ratio on CH₄ and ${\text{NO}}_3^{-}$/${\text{NO}}_2^{-}$ metabolism in the AME-D process, rather than the tendency of methane oxidation rates and denitrifying activities under different gaseous environments. The knowledge will allow a better understanding of the specific roles of the O₂:CH₄ ratio in CH₄ and ${\text{NO}}_3^{-}$/${\text{NO}}_2^{-}$ metabolism. It is expected that this will contribute to well-founded strategies that will improve nitrogen removal, one of bottle-necks in the application of the AME-D process.

In this study, the optimal O₂:CH₄ ratio for denitrification was found to be 0.25. At this point, denitrifying activity reached the highest level of 7.32 mmol ${\text{NO}}_2^{-}$-N/gVSS/d. When the O₂:CH₄ ratio was below the optimal ratio, nitrite removal was improved with the increased O₂:CH₄ ratio, presumably due to an increase in available substrates released by aerobic methanotrophs. Methanol, butyrate and formaldehyde were thermodynamically speculated as the main active intermediates of the AME-D process. When the O₂:CH₄ ratio was above the optimal ratio, nitrite removal was presumably inhibited by the excessive O₂. These results indicate that adjusting the O₂:CH₄ ratio can improve the cooperation between aerobic methanotrophs and denitrifiers to obtain better nitrogen removal performance using the AME-D process.

Author Contributions

JZ: contributed to the conception, experimental design, acquisition, analysis, and interpretation of data, and article drafting; XX, MY: analyzed and interpreted data; HW, ZM: contributed to data acquisition; WW: supervised the student and revised the article.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to express our appreciation to Dr. P. J. Strong from the Centre for Solid Waste Bioprocessing in the University of Queensland, Prof. Jiayang Cheng from Biological and Agricultural Engineering Department of North Carolina State University, Dr. Faqian sun, Dr. Cheng Wang, Dr. Pengfei Liu, PhD student Xingguo Han and Christina Horn for their help to improve the quality of this manuscript. We additionally thank the reviewers for their valuable suggestions. This work was supported by the China National Critical Project for Science and Technology on Water Pollution and Control under No. 2014ZX07101-012.

Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2017.01112/full#supplementary-material

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