Effect of methanol and glycerol on nitrous oxide (N2O) emission in two laboratory-scale modified Ludzak Ettinger (MLE) processes was investigated during three distinct periods: dissolved oxygen (DO) control by intermittent aeration with a DO controller, and high and low aeration rates. N2O consumption rate in an anoxic tank and aeration mode influenced N2O emission rates from the MLE processes. In the DO control period, N2O emission rate from the glycerol-fed MLE process was higher than the methanol-fed counterpart, likely caused by a higher N2O consumption rate in an anoxic tank of the methanol-fed process. During the period of a higher aeration rate, N2O emission rates from both processes were comparable. In contrast, during the period of a lower aeration rate, N2O emission rate from the methanol-fed MLE process was higher than that from the glycerol-fed counterpart likely because of a higher degree of nitrite accumulation, corroborated by statistical analysis. N2O consumption activities of biomasses fed with the different carbon sources were distinct. However, the high activity did not necessarily result in a decrease in N2O emission rate from an aerobic tank and the effect of nitrite on the emission was stronger under the tested conditions.

INTRODUCTION

Nitrous oxide (N2O) has currently attracted much more attention because of its high greenhouse effect, which is 300 times as high as that of carbon dioxide (CO2), and its dominant ozone-depleting effect (IPCC 2001; Ravishankara et al. 2009). Recently, it has been reported that the amount of N2O emission from municipal wastewater treatment plants (WWTPs) is not at a negligible level (IPCC 2007; Law et al. 2012). In particular, the amount is amplified in an anoxic/aerobic activated sludge system for nitrogen removal, i.e. a modified Ludzak Ettinger (MLE) process. In an MLE process, N2O is produced by incomplete denitrification, and an external carbon source is a driver to determine the amount of N2O production in an anoxic tank (Adouani et al. 2010; Hu et al. 2013a). The produced N2O is transferred to the ensuing aerobic tank where dissolved N2O is exhausted by aeration.

Heterotrophic denitrification reduces oxidized nitrogen compounds, i.e. nitrate and nitrite, to dinitrogen gas (N2) via nitric oxide (NO) and N2O (Knowles 1982), which require electron donors like external organic carbon. The addition of a carbon source is necessary to enhance denitrification at WWTPs for abidance with stringent effluent nitrogen limits. Methanol is generally used as an external electron donor for denitrification in WWTPs (Louzeiro et al. 2002). However, due to an increase in price (Masih et al. 2010) and safety concerns associated with the low flash-point, wastewater utilities may adopt an alternate external carbon source. Glycerol, a by-product of biodiesel production, is promising and the accessibility and availability is expectedly expanded (Bodík et al. 2009; Da Silva et al. 2009). The effect of other carbon sources, e.g. ethanol, glucose, sodium acetate and soluble starch, on N2O emission was reported (Adouani et al. 2010; Lu & Chandran 2010a; Hu et al. 2013a); however, there has been no report about N2O emission and consumption by glycerol as a carbon source.

An MLE process is widely employed in WWTPs subjected to investigation on N2O emission. Most previous studies focus on N2O emission from sequencing batch reactor systems (Hu et al. 2013a, b; Lu & Chandran 2010a; Lu et al. 2011) or on either merely nitrification or denitrification (Tsuneda et al. 2005). However, an MLE process has seldom attracted attention despite its importance toward mitigation of N2O emission. An anoxic tank in an MLE process receives mixed liquor from the ensuing aerobic tank, which brings dissolved oxygen (DO). This indicates that at a constant recycle ratio of mixed liquor from an aerobic tank in an MLE process, a redox condition in an anoxic tank varies depending on DO concentration in an aerobic tank. Reportedly, difference in the degree of nitrification, i.e. full nitrification vs partial nitrification, mainly caused by DO concentration control, may select microbial community structure (Lu & Chandran 2010a). Therefore, study on N2O emission from an MLE process fed with different carbon sources at different aeration regimes is of significance, geared toward mitigation of N2O emission in WWTPs.

The goal of this work was to analyse the effect of external carbon source, i.e. methanol and glycerol, on N2O emission in an MLE process at different DO concentrations by different aeration modes. To this end, the long-term operation of two laboratory-scale MLE processes fed with methanol and glycerol as an organic carbon was performed to infer N2O production and consumption.

MATERIALS AND METHODS

Reactor setup and operation

Two laboratory-scale MLE processes, comprised of anoxic (1 L), aerobic (3 L) and settling tanks (3 L), were continuously operated. Each process was fed with synthetic wastewater consisting of either methanol (System 1) or glycerol (System 2) as an external carbon source of 296 mg-C/L, and of 50 mg N/L, ensuring a carbon/nitrogen (C/N) ratio of approximately 6 to avoid unstable N2O production via incomplete denitrification (Law et al. 2012). The MLE processes were fed with activated sludge from a municipal WWTP (Kitatama-Ichigo, Fuchu, Tokyo). Hydraulic and solid retention times were set at 24 hours and 20 days, respectively. The internal recycle ratio of mixed liquor from an aerobic tank to an anoxic tank was 300% of the influent rate. DO concentration was lower than 0.1 mg/L in the anoxic tanks in both MLE systems. Three different aeration modes in an aerobic tank were applied: in Period 1 (day 23–98), DO concentration was controlled at 3.0 ± 0.5 mg/L by turning air on and off by a DO controller; in Period 2 (day 104–147), DO was controlled at 6.0 ± 0.5 mg/L at an aeration rate of 1.5 L/min; and in Period 3 (day 148–180), DO was controlled at 4.0 ± 0.5 mg/L at an aeration rate of 0.5 L/min. The reactor was halted from day 99 to 103 to recover from operational malfunction. pH and temperature were maintained at 7.5 ± 0.3 and 28 ± 2 °C respectively in both anoxic and aerobic tanks.

Analytical methods

The concentrations of nitrogen compounds, i.e. and were measured by an ion chromatography system (ICS-90, Dionex, Sunnyvale, USA), whereas total organic carbon (TOC) and total nitrogen (TN) concentrations were measured by a TOC analyser (TOC 5000A, Shimadzu, Kyoto, Japan). The concentrations of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) were measured according to the standard method (AWWA 1992). Gaseous and dissolved N2O concentrations were measured by a gas chromatograph with an electron capture detector (GC-ECD) (GC-14A, Shimadzu, Kyoto, Japan) and microelectrodes (Unisense, Aarhus, Denmark), respectively. An N2O gas emission rate was calculated using the following equation provided elsewhere (Tsuneda et al. 2005), with minor modification: 
formula
where is the N2O gas emission rate (g/min); Q is the gas flow rate during the aerobic phase (L/min); is the gaseous N2O concentration, obtained by collection of the exhausted gas (v/v) by a GC-ECD (Shimadzu, Kyoto, Japan); is the N2O molecular weight (44.02 g/mol); P is the atmospheric pressure (1 atm); R is the gas constant (0.082 L atm/K/mol) and T is the temperature (K).

N2O consumption rate by in-situ test

N2O consumption rate in an anoxic tank was evaluated based on respirometry on days 133 and 180. Pumps for the incoming wastewater and internal recycle were halted to avoid any DO coming to an anoxic tank. Subsequently, methanol and glycerol were fed to each anoxic tank in System 1 and System 2 to ensure TOC concentration of 600 mg/L. 100 mL of pure water sparged with 10% N2O gas for 10 min was added to each anoxic tank. This ensured dissolved N2O of 2800 μg N-N2O/L in the anoxic tank at the beginning. Dissolved N2O concentration was monitored on-line in each system by two individual dissolved N2O microelectrodes.

Statistical analysis

One-way analysis of variance (ANOVA) and t-test for significant difference analysis of each data point were employed. Pearson's correlation coefficient was chosen as a correlation analysis. These statistical analyses were performed by IBM SPSS Statistics for Windows, Version 20.0 (Armonk, NY: IBM Corp.).

RESULTS AND DISCUSSION

Nitrogen and TOC removal performance

Throughout the reactor operations for 180 days, the average MLSS concentrations were maintained at 1914 ± 270 mg/L and 2079 ± 480 mg/L in System 1 and System 2, respectively. The ratios of MLVSS/MLSS were 91.1 ± 5.0% and 91.6 ± 7.9% in System 1 and System 2 during the whole operational period. TOC and TN concentrations in each tank of Systems 1 and 2 are shown in Supplementary Figure S1 (available online at http://www.iwaponline.com/wst/072/250.pdf). TN removal efficiencies in Systems 1 and 2 were 95.8 ± 1.6% and 93.9 ± 3.1%, respectively. TOC removal efficiencies for the whole system in Systems 1 and 2 were 96.0 ± 1.2% and 94.8 ± 1.6%, respectively. This indicates that the effectiveness of glycerol as an external carbon source was comparable with that of methanol and both systems achieved stable TOC and TN removal efficiencies. Denitrification efficiencies in the anoxic tanks of Systems 1 and 2 were 99.6 ± 1.3% and 99.8 ± 0.8%, respectively.

Performance of N2O emission rate from both systems by different aeration modes

The average N2O emission rates from System 1 and System 2 during Periods 1, 2 and 3 are shown in Figure 1. N2O emission rates from both systems were different in each period. N2O-N conversion ratios in Systems 1 and 2 during the entire periods, obtained from the quantity of N2O production over removed nitrogen, ranged from 0.33% to 4.33% and from 0.34% to 5.52%, respectively. The ratios were lower than those of an anoxic/aerobic sequencing batch reactor (Hu et al. 2013b) and a batch reactor for denitrification (Adouani et al. 2010) where acetate was an external carbon source.

Figure 1

Box plot of N2O emission rates from System 1 and System 2 during three different periods. The boundaries of the box closest to and farthest from zero indicate the 25th and 75th percentiles, respectively, the line within the box marks the median, and the solid dot inside the box indicates the mean. The error bar above and below each box indicates the 90th and 10th percentiles, respectively, and the crosses indicate outlying points. Different letters above the bars indicate significant differences (P < 0.01) between N2O emission rates.

Figure 1

Box plot of N2O emission rates from System 1 and System 2 during three different periods. The boundaries of the box closest to and farthest from zero indicate the 25th and 75th percentiles, respectively, the line within the box marks the median, and the solid dot inside the box indicates the mean. The error bar above and below each box indicates the 90th and 10th percentiles, respectively, and the crosses indicate outlying points. Different letters above the bars indicate significant differences (P < 0.01) between N2O emission rates.

In Period 1, average N2O emission rates from System 1 and System 2 were 0.76 μg N-N2O/min and 1.79 μg N-N2O/min with average N2O-N conversion ratios of 0.71% and 1.74%, respectively. The differences in the N2O emission rates and conversion ratios were statistically significant (P < 0.001 and P < 0.001, ANOVA). In Period 2, average N2O emission rates from System 1 and System 2 were 1.75 μg N-N2O/min and 2.13 μg N-N2O/min with N2O-N conversion ratios of 1.56% and 2.07%, respectively. The rates and ratios in this period were not statistically significant (P = 0.219 and P = 0.238, ANOVA). In Period 3, average N2O emission rates from Systems 1 and 2 were 3.21 μg N-N2O/min and 1.65 μg N-N2O/min with N2O-N conversion ratios of 2.91% and 1.82%, respectively. The N2O emission rates and conversion ratios were statistically significant (P < 0.001 and P < 0.05, ANOVA). The trends of N2O emission rates and conversion ratios were the opposite in Periods 1 and 3: a higher amount of N2O was produced in the glycerol-fed system than in the methanol-fed system in Period 1, whereas the opposite trend was obtained in Period 3. The reason for this is putatively due to compositions of nitrogen constituents and different microbial community structure, which warrants future investigation. Despite the point to be elucidated, these results suggest that not only aeration modes but also organic carbon source determined N2O emission.

Performance of dissolved N2O concentration in both systems by different aeration modes

Dissolved N2O concentration characteristics

As shown in Figure 2, average dissolved N2O concentrations in the anoxic tanks of System 1 and System 2 in Period 1 were 16.80 μg N-N2O/L and 29.96 μg N-N2O/L, respectively, which shows statistically significant difference (P = 0.002, ANOVA). Average dissolved N2O concentrations in the aerobic tanks of System 1 and System 2 were 37.24 μg N-N2O/L and 46.76 μg N-N2O/L, respectively, showing statistically insignificant values (P = 0.455, ANOVA). In Period 2, average dissolved N2O concentrations in the anoxic tanks of System 1 and System 2 were 31.36 μg N-N2O/L and 21.56 μg N-N2O/L, respectively (P = 0.093, ANOVA). Average dissolved N2O concentrations in the aerobic tanks of System 1 and System 2 were 11.92 μg N-N2O/L and 33.59 μg N-N2O/L, respectively, with statistical significance (P = 0.046, ANOVA). In Period 3, average dissolved N2O concentrations in the anoxic tanks of System 1 and System 2 were 40.32 μg N-N2O/L and 56.28 μg N-N2O/L, respectively, with statistical significance (P = 0.044, ANOVA). On the contrary, average dissolved N2O concentrations in the aerobic tanks of System 1 and System 2 were 29.40 and 42.00 μg N-N2O/L, respectively, with no statistical significance (P = 0.077, ANOVA). These results indicate that the ranges in dissolved N2O concentrations in anoxic and aerobic tanks fed with different organic carbon sources were broad. Furthermore, N2O concentrations in the aerobic and anoxic tanks did not necessarily correlate with N2O emission rates as shown in Figures 1 and 2. For instance, although a higher N2O concentration in the anoxic tank during Period 3 was observed in System 2, a lower amount of N2O was emitted from the aerobic tank in System 2. This inconsistent result underscores that N2O emission rate in an MLE process is not simply caused by dissolved N2O concentration but by other operational factors as well.

Figure 2

Box plot of dissolved N2O concentration in the (a) anoxic and (b) aerobic tanks of Systems 1 and 2 during three different periods. The crosses indicate outlying points. Different letters above the bars indicate significant differences (P < 0.05) between dissolved N2O concentration.

Figure 2

Box plot of dissolved N2O concentration in the (a) anoxic and (b) aerobic tanks of Systems 1 and 2 during three different periods. The crosses indicate outlying points. Different letters above the bars indicate significant differences (P < 0.05) between dissolved N2O concentration.

Effect of aeration modes on dissolved N2O concentration

Periods 1 to 3 employed different aeration modes. Intermittent aeration in Period 1 can avoid less oxygen transfer from an aerobic tank to an anoxic tank where N2O reducing bacteria potentially consume dissolved N2O. This should lead to a lower dissolved N2O concentration in an anoxic tank. This trend was observed in System 1, receiving a lower N2O concentration (Figure 2). In Periods 2 and 3, continuous aeration provided higher flux of oxygen into the anoxic tank, plausibly resulting in a lower N2O consumption rate in an anoxic tank. A higher dissolved N2O concentration in the anoxic tank was detected especially in Period 3 (Figure 2). The trend infers that N2O concentration was not sufficiently consumed in the anoxic tank especially in Period 3 and, therefore, the remaining N2O was transferred to the aeration tank, resulting in N2O stripping to the gas phase.

Different aeration rates employed in Periods 2 and 3 permitted different DO concentrations. In Period 2, an excessive aeration rate resulted in high DO concentration (6.0 ± 0.5 mg/L), which negatively affected N2O consumption rate in the anoxic tank. However, this does not explain the result shown in Figure 1 where N2O emission rates were comparable in System 2 in Periods 2 and 3. A clear rationale to increase or decrease N2O emission rates in our experiments warrants more investigation.

Relationship between dissolved N2O concentration and N2O emission rate

The correlation between each nitrogen constituent and N2O concentration as well as emission rate are shown in Table 1. Nitrite concentration in the aerobic tank and effluent was positively correlated with N2O emission rate in Systems 1 and 2 in all the periods. This is consistent with previous literature, reporting nitrite as a driver for N2O emission (Colliver & Stephenson 2000; Kampschreur et al. 2009; Foley et al. 2010). As shown in Supplementary Figure S2 (available online at http://www.iwaponline.com/wst/072/250.pdf), TN concentrations were comparable between System 1 and System 2 in the aerobic tank in each period, but nitrite concentrations were different in Periods 1 and 3. Irrespective of the carbon sources, higher nitrite concentration of one system compared with the other, i.e. System 2 in Period 1 and System 1 in Period 3, resulted in higher N2O concentration and N2O emission rate in and from the aerobic tank, respectively. High sensitivity of nitrite toward N2O production is reported elsewhere where nitrite pulse of 10 mg N/L leads to a four- to eight-fold increase in N2O emission during nitrification (Tallec et al. 2006), the trend of which tallies with our observation. As mentioned before, the difference between dissolved N2O concentrations in the anoxic tank of Systems 1 and 2 was not in concordance with N2O emission rate in Period 3. Therefore, N2O production and stripping from the liquid to the gaseous phase in an aerobic tank was a main cause to lead to N2O emission. Nitrate concentration in the aerobic tank and effluent were negatively correlated with N2O concentration in Systems 1 and 2 (P < 0.05). The correlation matrices as shown in Table 1 corroborate that nitrite triggers N2O production in the aerobic tank irrespective of the carbon sources, resulting in high N2O emission rate.

Table 1

Correlation matrices of water quality, N2O gas concentration and N2O emission rate (n = 41–44)

Variable Average concentrations [mg N/L]
 
Pearson's correlation coefficient
 
System 1 System 2 System 1
 
System 2
 
N2O gas concentration N2O gas emission rate N2O gas concentration N2O gas emission rate 
(AN) 13.6 ± 3.6 12.8 ± 3.9 −0.257 −0.077 0.037 −0.097 
(AER) 1.2 ± 1.5 1.8 ± 2.0 0.539** 0.499** 0.380* 0.379* 
(AER) 4.8 ± 2.0 4.3 ± 1.9 −0.457** −0.218 −0.467** −0.348* 
(effluent) 0.2 ± 0.4 0.4 ± 0.7 0.567** 0.489** 0.378* 0.537** 
(effluent) 2.1 ± 1.2 2.3 ± 1.6 −0.379* −0.168 −0.500** −0.278 
TN loss (AN) 6.3 ± 1.8 7.9 ± 1.0 0.138 0.161 0.182 0.068 
TN loss (AER) 3.6 ± 1.0 2.0 ± 0.9 −0.064 −0.174 −0.118 −0.102 
Variable Average concentrations [mg N/L]
 
Pearson's correlation coefficient
 
System 1 System 2 System 1
 
System 2
 
N2O gas concentration N2O gas emission rate N2O gas concentration N2O gas emission rate 
(AN) 13.6 ± 3.6 12.8 ± 3.9 −0.257 −0.077 0.037 −0.097 
(AER) 1.2 ± 1.5 1.8 ± 2.0 0.539** 0.499** 0.380* 0.379* 
(AER) 4.8 ± 2.0 4.3 ± 1.9 −0.457** −0.218 −0.467** −0.348* 
(effluent) 0.2 ± 0.4 0.4 ± 0.7 0.567** 0.489** 0.378* 0.537** 
(effluent) 2.1 ± 1.2 2.3 ± 1.6 −0.379* −0.168 −0.500** −0.278 
TN loss (AN) 6.3 ± 1.8 7.9 ± 1.0 0.138 0.161 0.182 0.068 
TN loss (AER) 3.6 ± 1.0 2.0 ± 0.9 −0.064 −0.174 −0.118 −0.102 

The abbreviations AN and AER represent an anoxic and aerobic tank, respectively.

*P < 0.05.

**P < 0.01.

N2O consumption rate in an anoxic tank by in-situ activity test

N2O consumption rates in an anoxic tank are summarized in Figure 3. Specific N2O consumption rates were estimated from a decline of dissolved N2O concentration. On day 133 (Period 2), N2O consumption rates in Systems 1 and 2 were 4.73 mg N-N2O/g MLVSS/hour and 3.74 mg N-N2O/g MLVSS/hour, respectively. On day 180 (Period 3), N2O consumption rates in Systems 1 and 2 were 6.25 mg N-N2O/g MLVSS/hour and 3.26 mg N-N2O/g MLVSS/hour, respectively. N2O consumption rates in System 1 were higher than System 2 on both days, which suggests that a specific N2O consumption rate is not necessarily a determinant factor for N2O emission rate from an aerobic tank as shown in Figure 1. Reduction of an aeration rate from Period 2 to 3 increased a specific N2O consumption rate in System 1 but not in System 2. Given that N2O reductase, an enzyme to reduce N2O to N2, was susceptible to oxygen especially among denitrification enzymes (Zumft 1997), the decrease in an aeration rate reduced oxygen flux recycled from the aerobic to anoxic tank, resulting in an increase in N2O consumption rate in System 1 fed with methanol. This propensity was not observed in System 2 fed with glycerol, showing a constant N2O reduction rate irrespective of the different aeration rates. The reason for this is not clear; however, different microbial community structure may explain this inconsistent result. Reportedly, an external organic carbon source provides a stronger impact on denitrifying community structure than other factors, leading to different performance of N2O production and consumption by phylogenetically and physiologically distinct denitrifying bacteria (Baytshtok et al. 2008, 2009; Hagman et al. 2008; Xia et al. 2008; Lu & Chandran 2010b; Wan et al. 2011). Although a higher N2O consumption rate was observed in System 1 (Figure 3), the system emitted N2O at a higher rate than System 2 from the aerobic tank in Period 3 (Figure 1), underpinning that N2O emission rate increased because of nitrite accumulation under the tested conditions.

Figure 3

N2O consumption rate in the anoxic tank of Systems 1 and 2 on days 133 and 180.

Figure 3

N2O consumption rate in the anoxic tank of Systems 1 and 2 on days 133 and 180.

The different N2O consumption rate and nitrogen removal performance were putatively related to microbial community structure fed with different carbon sources, according to the previous study (Lu et al. 2014). Characterization of microbial ecology in sequencing batch reactors (SBRs) fed with methanol and glycerol as electron donors has illuminated that the different genera, Methyloversatilis spp. and Hyphomicrobium spp. in the methanol-fed SBR, and Citrobacter spp. in the glycerol-fed SBR, are predominant (Lu et al. 2011). Reportedly, Hyphomicrobium spp. hold N2O reductase (Yamaguchi et al. 2003) and contribute to mitigation of N2O emission from a bioreactor for swine wastewater treatment (Yamashita et al. 2014). In contrast, Citrobacter spp. trigger nitrite accumulation and N2O production in soil (Smith & Zimmerman 1981; Bleakley & Tiedje 1982). Although analysis of microbial community was not conducted in this study, the higher N2O reduction rate in the methanol-fed MLE as shown in Figure 3 may be caused by abundance of highly active N2O reducing bacteria. The analysis of microbial community structure warrants future investigation.

CONCLUSIONS

This study revealed that MLE processes fed with methanol and glycerol achieved comparable TN and TOC removal efficiencies, indicating that glycerol can be a promising alternate as an electron donor. Carbon source resulted in a different N2O consumption rate in an anoxic tank, which may indirectly influence N2O emission from an MLE process. An intrinsic N2O consumption rate under an anoxic condition in the methanol-fed MLE process was higher than that in the glycerol-fed MLE process. However, the higher N2O consumption activity did not dominantly contribute to mitigation of N2O emission from an aeration tank under the tested conditions. The statistical analysis demonstrated that gaseous N2O concentration and emission rate in an aerobic tank were boosted mainly by nitrite concentration.

ACKNOWLEDGEMENTS

This work was financially supported in part by Grant-in-Aid for Scientific Research (no. 268245 and 26701009) from the Japan Society for the Promotion of Science and the New Energy and Industrial Technology Development Organization (11B13001d). The authors thank Ms Kanako Mori (Tokyo University of Agriculture and Technology) for technical assistance.

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Supplementary data