影响细菌反硝化法测量硝酸盐氧同位素的因素分析

Factors influencing the accuracy of the denitrifier method for determining the oxygen isotopic composition of nitrate 影响细菌反硝化法测量硝酸盐氧同位素的因素分析 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:jcshi@zju.edu.cn †E-mail:jcshi@zju.edu.cn jcshi@zju.edu.cn 1 2 Institute of Soil and Water Resources and Environmental Science, College of Environmental and Resource Sciences, Zhejiang Provincial Key Laboratory of Agricultural and Environment, Zhejiang University, Hangzhou 310058, China Institute of Soil and Water Resources and Environmental Science, College of Environmental and Resource Sciences, Zhejiang Provincial Key Laboratory of Agricultural and Environment, Zhejiang University, Hangzhou 310058, China Department of Environmental Sciences, University of California, Riverside, CA 92521, USA Department of Environmental Sciences, University of California, Riverside, CA 92521, USA The denitrifier method is widely used as a novel pretreatment method for the determination of nitrogen and oxygen isotope ratios as it can provide quantitative and high-sensitivity measurements. Nevertheless, the method is limited by relatively low measurement accuracy for δ 18 O. In this study, we analyzed the factors influencing the accuracy of δ 18 O determination, and then systematically investigated the effects of dissolved oxygen concentrations and nitrate sample sizes on estimates of the δ 15 N and δ 18 O of nitrate reference materials. The δ 18 O contraction ratio was used to represent the relationship between the measured difference and true difference between two reference materials. We obtained the following main results: (1) a gas-liquid ratio of 3:10 (v/v) in ordinary triangular flasks and a shaking speed of 120 r/min produced an optimal range (1.9 to 2.6 mg/L) in the concentration of dissolved oxygen for accurately determining δ 18 O, and (2) the δ 18 O contraction ratio decreased as nitrate sample size decreased within a certain range (1.0 to 0.1 μmol). Our results suggested that δ 18 O contraction is influenced mainly by dissolved oxygen concentrations in pure culture, and provided a model for improving the accuracy of oxygen isotope analysis. 目的 2 针对硝酸盐氮氧同位素新型预处理方法--细菌反硝化法尚存在的氧同位素测试精度低和两种标准物质间的δ 18 O差值会收缩等问题进行优化试验和机理探究. 创新点 2 首次深入探究纯培养时的溶解氧浓度和硝酸盐进样量对同位素测试结果的影响及原因,可为氧同位素测试精度的提高提供方法和参考. 方法 2 通过控制培养瓶内的气液比(1:5、3:10、2:5和3:5)和摇床转速(60和120 r/min)来调节纯培养时的溶解氧浓度,并设计不同的硝酸盐进样量(0.05~1.00 µmol),由硝酸盐标准物质的测试结果判断适宜的溶解氧浓度和硝酸盐进样量. 结论 2 标准物质间的δ 18 O差值收缩主要受溶解氧浓度的影响.根据氧同位素测试结果的精度和δ 18 O差值收缩率的大小,得出以下结论:当普通三角瓶内的气液比为3:10,摇床转速为120 r/min时的溶解氧浓度最佳(1.9~2.6 mg/L),0.2~0.4 nmol的硝酸盐进样量最适宜. Denitrifier method Nitrate δ 15 N δ 18 O Dissolved oxygen δ 18 O contraction 细菌反硝化法 硝酸盐 δ 15 N δ 18 O 溶解氧 δ 18 O收缩 Introduction 1 Natural abundance stable isotope signatures have been successfully used in numerous surface water and groundwater systems to trace contamination sources (Widory et al., 2005 2005 ; Popescu et al., 2015 2015 ). The isotopic composition of nitrate (δ 15 N and δ 18 O) can provide valuable information to characterize the dominant sources of nitrate pollution and quantitatively estimate the contributions from different sources by applying stable isotope mixing models (e.g. SIAR) (Oelmann et al., 2007 2007 ; Ward et al., 2010 2010 ; Rock et al., 2011 2011 ; Matiatos, 2016 2016 ). A simple, rapid, low-contamination, cost-effective, and highly precise sample preparation method for isotope measurement is urgently needed. Continuous-flow isotope ratio mass spectrometry (IRMS) requires nitrate in water to be converted to a solid (e.g. ammonium salt or AgNO 3 ) and then to a gas (e.g. N 2 and CO or N 2 and CO 2 ). Compared with other preparation methods like ion exchange chromatography (Silva et al., 2000 2000 ), the diffusion method (Stark and Hart, 1996 1996 ), and chemical method (Stevens and Laughlin, 1994 1994 ), the denitrifier method has the advantage of simultaneously measuring nitrogen and oxygen isotopes of nitrate at concentrations as low as 0.1 μmol/L and in sample sizes of less than 10 ml (Casciotti et al., 2002 2002 ). In addition, the denitrifier method can effectively reduce errors originating from atmospheric leaks, problems associated with reduction of NO 3 − to NH 4 + , or contamination from other N-sources (Christensen and Tiedje, 1988 1988 ). The denitrifier method was first introduced by Sigman et al. ( 2001 2001 ) as a novel method to measure the natural abundance of nitrate isotopes in seawater and freshwater. It was based on the isotopic analysis of nitrous oxide (N 2 O) generated from nitrate by denitrifying bacteria (e.g. Pseudomonas aureofaciens and Pseudomonas chlororaphis ) lacking N 2 O-reductase activity. Materials and methods like autosampler modification, water sample treatment, pre-concentration to denitrify cultures, and NO 2 − removal are being constantly updated to improve the performance of the denitrifier method by increasing the precision and sensitivity of isotopic analysis (Mørkved et al., 2007 2007 ; Granger and Sigman, 2009 2009 ; McIlvin and Casciotti, 2011 2011 ; Weigand et al., 2016 2016 ). Moreover, the application of the denitrifier method is continuously expanding, and it is now used in the fields of botany, soil science, atmospheric science, and the study of the global nitrogen cycle (Dahal and Hastings, 2016 2016 ). Presently, the measurement precision of the oxygen isotope (0.5‰ to 2.0‰) is far lower than that of the nitrogen isotope (0.20‰ to 0.46‰) in nitrate dissolved in water samples (Vicars et al., 2013 2013 ). This is because the conversion of nitrate to N 2 O maintains a mass balance reaction for nitrogen but not for oxygen. Isotope fractionation and the exchange of oxygen atoms with water may lead to δ 18 O in N 2 O differing from its original nitrate. Experiments with 18 O-labeled water were conducted by Casciotti et al. ( 2002 2002 ). These experiments indicated that oxygen atoms exchanged with water accounted for less than 10% of those in the N 2 O products for P . aureofaciens . In addition, both oxygen isotope fractionation and oxygen atom exchange should be consistent within a given batch of analyses. Accordingly, the effects of isotopic fractionation, exchange, and a blank on δ 18 O measurements of water samples were accurately quantified by analyzing two reference materials (RMs) of known δ 18 O within the same batch. Nonetheless, due to incomplete oxygen conversion and an unclear reaction process for oxygen isotope exchange, the calibration of the oxygen isotope was complicated and the measurement precision was low (Dai et al., 2017 2017 ). Consequently, the limitation of the denitrifier method was the difficulty in accurately determining the δ 18 O of nitrate. In addition, there was a contraction in the oxygen isotopic difference between IAEA-NO-3 (International Atomic Energy Agency, Vienna, Austria, subsequently referred to as “N3”) and USUG34 (the National Institute of Standards and Technology, Gaithersburg, MD, USA) (Weigand et al., 2016 2016 ). It is known that the denitrification process is influenced by the dissolved oxygen (DO) concentration, carbon source, nitrate concentration, pH, and temperature (Deng, 2010 2010 ). In addition, the experimental bacterium in the denitrifier method is an aerobic denitrifying bacterium. Therefore, the effect of DO and nitrate concentration on δ 18 O of N 2 O formed by denitrification should be noted when pH (neutral), temperature (26 °C), and carbon source are constant. In a preliminary experiment, we found that the δ 18 O of N 2 O derived from nitrate varied with the headspace in culture bottles, as noted by Xu et al. ( 2012 2012 ). Therefore, the objectives of this study were: (1) to examine the effects of DO concentrations and nitrate sample sizes on the precision of isotopic analysis, especially for δ 18 O; (2) to explore the cause of poor analytical precision and the contraction of δ 18 O; and (3) to identify the optimal DO concentrations and nitrate sample sizes to avoid δ 18 O contraction. Materials and methods 1 Materials 2 P . aureofaciens (strain number: ATCC 13985; a facultative anaerobic bacterium, recently reclassified as a strain of P . chlororaphis ) was selected as the experimental bacterium in this study. The nitrate isotopic RMs IAEA-NO-3, USGS32, and USGS34 were used to evaluate the analytical methods according to the accuracy and precision of measurement results (Hastings et al., 2003 2003 ; Weigand et al., 2016 2016 ). The isotopic composition of the RMs is shown in Table 1 1 . Uncertainties for δ 15 N, δ 18 O, or Δ 17 O are two times the standard uncertainty of reported values from a single study (Böhlke et al., 2003 2003 ). Data sources (Böhlke and Coplen, 1995 1995 ; Böhlke et al., 2003 2003 ). Data sources (Böhlke and Coplen, 1995 1995 ; Böhlke et al., 2003 2003 ; Qi et al., 2003 2003 ). Data source (Böhlke et al., 2003 2003 ) 2 Reference material 2 15 N isotope ratio (×10 3 ) 2 18 O isotope ratio (×10 3 ) 2 17 O isotope ratio (×10 3 ) δ 15 N U a a δ 18 O U a a Δ 17 O U a a  IAEA-NO-3 b b 4.7 ±0.3 25.6 ±0.4 −0.2 ±0.2  USGS32 c c 180.0 Exact 25.7 ±0.4  USGS34 d d −1.8 ±0.2 −27.9 ±0.6 −0.1 ±0.2 Method set-up 2 Based on the previously published method (Sigman et al., 2001 2001 ; Mørkved et al., 2007 2007 ; McIlvin and Casciotti, 2011 2011 ), we focused on the oxygen isotopic ratios of nitrate. We divided the procedures (Fig. 1 1 ) of the denitrifier method into the following steps: (1) resuscitation of the denitrifying strains, (2) pure culture, (3) test bottle-making, (4) sample pretreatment and nitrate conversion, (5) N 2 O extraction and isolation, and (6) measurement and calibration of isotopic ratios. First, P . aureofaciens stored in a −80 °C refrigerator was revived by daubing an agar plate. Second, a 5-ml starter was prepared from single colonies to generate inoculum overnight. The starter was transferred to a culture flask (routine lab equipment, triangular flask), sealed with a rubber stopper and Parafilm® M (#996, Pechiney Plastic Packaging, Menasha, WI, USA), and incubated using amended medium with KNO 3 for 7 d. Third, the medium was concentrated 10-fold (cell slurry was dissolved using fresh amended medium without KNO 3 after centrifuging) and sealed in a 20-ml headspace vial (2 to 3 ml per vial) with butyl rubber stoppers (Ht-0942, Qingdao Hiprove Medical Technologies Co., Ltd., Qingdao, China). The headspace vial was used as the test bottle after purging for 2–3 h using helium or high purity nitrogen to remove N 2 O and ensure anaerobic conditions. Next, water samples were heated in a water bath for 1 h at 80 °C (Mørkved et al., 2007 2007 ) to inhibit the activity of native denitrifying bacteria. The pH was adjusted to 3.5 using ascorbate to eliminate the disturbance of nitrite (Xu et al., 2012 2012 ), and to neutral using NaOH solution. After samples (less than 10 ml) containing 0.2 to 0.4 μmol nitrate were injected into the test bottles overnight for NO 3 − conversion, 0.1 to 0.2 ml of 10 mol/L NaOH was used to lyse the bacteria and scrub any CO 2 gas in each test bottle. Next, N 2 O was extracted from the test bottles off-line and injected into a Thermo Scientific pre-concentrator (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) to be isolated on-line. N 2 O purity was analyzed using IRMS (Thermo Finnigan MAT 253). Finally, the RMs were processed with the samples simultaneously. The nitrate concentrations of the RMs were matched to those of the samples, and the data were used to correct the nonlinearity of the mass spectrometer and blanks associated with the procedure. The natural abundances of 15 N and 18 O were calculated as δ 15 N and δ 18 O, respectively, expressed in per mill units (‰): natural abundance=[( R sample / R standard )−1]×1000‰, where R sample and R standard are the ratios of heavy isotope to light isotope of sample and standard, respectively (Liu et al., 2012 2012 ). The standard for 15 N was air, and for 18 O was the Vienna Standard Means Ocean Water (VSMOW). To facilitate analysis, we introduced the contraction ratio ( C ) to reflect the reliability of measurement results: C =( T − M )/ T ×100%, where M and T are the measured and true δ difference between RMs, respectively. Accordingly, the result is more accurate when the contraction ratio is smaller. Experiment I: effect of DO concentrations in pure culture on δ 18 O measurement 2 The DO concentrations in medium can be affected by both gas-liquid ratios in the culture bottle and shaking speeds. To explore the optimal DO concentrations in ordinary triangular flasks and the relationship between DO concentrations in pure culture and the analytical precision of δ 18 O, we first conducted an experiment under both non-sealed and sealed conditions. Generally, the ratio of gas to liquid was about 1:5 (v/v) for the culture bottle (Xu et al., 2012 2012 ). The volume of the non-sealed pure culture with medium accounted for 5/6 of the actual volume (600 ml) of the 500 ml triangular flasks. The sealed conditions provided limited volume for the culture medium (375, 428, and 500 ml) in the flasks when other conditions were consistent with previously published methods (Sigman et al., 2001 2001 ; Mørkved et al., 2007 2007 ). For non-sealed cultures, the top of each culture flask was wrapped with six layers of gauze and four layers of newspaper. For airtight flasks, rubber stoppers and Parafilm M were used to maintain predefined gas-liquid ratios (3:5, 2:5, and 1:5; v/v). After the optimal gas-liquid ratios in culture flasks were determined, two shaking speeds (60 and 120 r/min) were selected based on the literature (Sigman et al., 2001 2001 ; McIlvin and Casciotti, 2011 2011 ). Analytical pure (AR) KNO 3 and two RMs (USGS34 and N3) were converted to N 2 O using denitrifying culture to evaluate the optimal gas-liquid ratio and shaking speed. Experiment II: effect of nitrate sample sizes on δ 18 O measurement 2 The denitrifier method has been shown to have high sensitivity (tested down to 0.01 μmol nitrate) (Casciotti et al., 2002 2002 ). To examine the effect of nitrate sample size on 18 O measurement, the RM (N3 and USGS34) solutions were diluted across a gradient of nitrate concentrations (0.025 to 0.500 μmol/L), and then 2 ml of the diluted solution was injected into the test bottle to be denitrified in 2.5 ml of experimental bacteria solution. The analytical precision and contraction ratios of δ 15 N and δ 18 O in RMs were measured for different nitrate sample sizes. Results 1 Non-sealed cultures 2 The average standard deviations (SDs) ( n =6) of δ 15 N were 0.25‰ for KNO 3 (AR), 0.28‰ for N3, and 0.57‰ for USGS34. The difference (6.38‰) between measured values of δ 15 N for USGS34 and N3 was close to the true difference of 6.5‰. However, the observed variation of measured δ 18 O values (the maximum SD=3.42‰) was higher than that of δ 15 N. The difference for measured δ 18 O values between the two RMs ranged from 0.63‰ to 4.90‰, which was much lower than the true difference of 53.5‰. Moreover, the δ 18 O contraction ratios were higher than 90% in non-sealed conditions. Sealed cultures with different gas-liquid ratios 2 The measured δ 18 O values of RMs under sealed-culture conditions with different gas-liquid ratios are shown in Fig. 2 2 . The results indicated that the measured δ 18 O differences between USGS34 and N3 increased from 7.72‰ to 47.98‰, and δ 18 O contraction ratios declined from 85.6% to 10.3% when the gas-liquid ratios decreased from 3:5 to 1:5 (data not shown). The contraction ratios were higher than 85.4% under the gas-liquid ratios of 3:5 and 2:5. N 2 O sometimes could not be detected using IRMS when the gas-liquid ratio was 1:5 with a low cell density (optical density (OD)=0.19, while OD=0.31 for a gas-liquid ratio of 2:5). To harvest sufficient cells and guarantee complete nitrate conversion in water samples, the gas-liquid ratio in the triangular flasks needed to be greater than 1:5. Therefore, the gas-liquid ratio of 3:10 (v/v) was selected for our continuous experiments, and the measurement accuracies of δ 15 N and δ 18 O were significantly improved. The average SD values were reduced to as low as 0.11‰ (N3) and 0.16‰ (USGS34) for δ 15 N, and 0.20‰ (N3) and 0.22‰ (USGS34) for δ 18 O. The δ 18 O contraction ratios ranged from 4.9% to 7.3% with a mean value of 6.2%. Nitrate sample sizes 2 Samples with different nitrate sample sizes were analyzed based on the case of a gas-liquid ratio of 3:10 (v/v) (Table 2 2 ). The measurement precision of δ 18 O for either high or low NO 3 − sample sizes was lower than that for medium nitrate sample sizes in RMs (i.e. 0.2 and 0.4 μmol NO 3 − , P < 0.05). When the nitrate sample size was 0.05 μmol, the contraction ratios of δ 15 N and δ 18 O were higher than those of other nitrate sample sizes. When the nitrate sample sizes ranged from 0.2 to 0.4 μmol, the measured differences between USGS34 and N3 were close to the true difference values, and δ 18 O contraction ratios were less than the average 6.2% (Table 2 2 ). RM: reference material; SD: standard deviation; C : contraction ratio 2 No. of batches 2 RM 2 Nitrate sample size (μmol) 2 δ 15 N (‰) 2 δ 18 O (‰) 2 Difference (‰) 2 C (%) Mean SD Mean SD δ 15 N δ 18 O δ 15 N δ 18 O 2  1 N3 2 1.00 4.59 0.08 21.57 0.27 2 6.41 2 49.62 2 1.4 2 7.3 USGS34 −1.82 0.07 −28.05 0.33 2  2 N3 2 0.40 4.64 0.03 22.14 0.12 2 6.44 2 50.02 2 0.9 2 6.5 USGS34 −1.80 0.19 −27.87 0.07 2  3 N3 2 0.20 4.68 0.08 22.48 0.06 2 6.47 2 50.32 2 0.4 2 6.0 USGS34 −1.82 0.24 −27.84 0.12 2  4 N3 2 0.20 4.68 0.06 22.29 0.21 2 6.49 2 50.21 2 0.2 2 6.2 USGS34 −1.80 0.23 −27.91 0.20 2  5 N3 2 0.10 4.68 0.09 23.07 0.18 2 6.46 2 50.87 2 0.7 2 4.9 USGS34 −1.78 0.15 −27.81 0.28 2  6 N3 2 0.05 4.54 0.29 22.32 0.36 2 6.20 2 50.12 2 4.7 2 6.3 USGS34 −1.66 0.07 −27.80 0.30 Discussion 1 Effect of the nitrate sample sizes on δ 18 O measurement 2 The measurement accuracies of δ 15 N and δ 18 O were significantly improved using the gas-liquid ratio of 3:10, and the δ 18 O contraction ratios decreased as nitrate sample sizes decreased (except for 0.05 μmol). These results agree with the observations of Weigand et al. ( 2016 2016 ). When the nitrate sample size was 0.05 μmol, it was significantly affected by the blank, resulting in a high contraction ratio for both δ 15 N and δ 18 O. Although the optimal nitrate sample sizes reduced δ 18 O contraction ratios, there was still a slight contraction (6.0% to 6.5%) in the measured δ 18 O values (Table 2 2 ). Weigand et al. ( 2016 2016 ) suggested that a change in nitrate sample sizes was not the sole cause of the δ 18 O contraction. Compared with nitrogen isotopes, oxygen isotopes are more difficult to measure due to the exchange of oxygen atoms with water, isotope fractionation, blank, and other factors. The nitrate in water samples can be completely converted to N 2 O in a high cell density bacterial solution, and thus no nitrogen isotope fractionation occurs according to mass balance. However, this is not the case for oxygen atoms. Xue et al. ( 2010 2010 ) noted that only one of the six oxygen atoms in the initial nitrate pool was represented in the N 2 O (2NO 3 − → N 2 O) analysis (Fig. 3 3 ). Moreover, the intermediates (nitrite and nitric oxide; Fig. 3 3 ) during the conversion would exchange oxygen atoms with water (Ye et al., 1991 1991 ). The degree of exchange may be related to the type of nitrite reductase. The copper-type nitrite reductase of P . aureofaciens incorporates fewer oxygen atoms from water into N 2 O than the heme-type nitrite reductase of P. chlororaphis (Glockner et al., 1993 1993 ). That is, not all oxygen atoms in N 2 O are derived from the nitrate pool, and this issue can lead to uncertainty in isotopic analysis using the denitrifier method. Nar, Nir, Nor, and Nos represent nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively (Morozkina and Zvyagilskaya, 2007 2007 ). The gray shading in the figure indicates a process that does not occur Moreover, there was a preferential loss of 16 O in these reactions that caused a difference in δ 18 O between nitrate and the product N 2 O, even if the conversion of nitrate to N 2 O was complete (Casciotti et al., 2002 2002 ). The denitrifying medium may contain trace amounts of NO 2 − , NO, and N 2 O generated from previous pure cultures. Previous studies showed that even if fresh medium was used instead of spent medium to re-suspend the cell slurry, and the fresh bacterial solution was purged by helium for more than 4 h, the denitrifying medium still resulted in lower blanks (about 0.04 nmol of N) (McIlvin and Casciotti, 2011 2011 ). In addition, the δ 18 O contraction of RMs may be affected by addition of KH 2 PO 4 or K 2 HPO 4 , the residence time of nitrite in the bacterial solution (Weigand et al., 2016 2016 ), and the off-line headspace sampling (Mørkved et al., 2007 2007 ). Effect of the DO concentrations on δ 18 O measurement 2 Ideally, the factors (e.g. oxygen atom exchange with water, oxygen isotope fractionation, and blank size) can be eliminated by adding RMs to a batch of samples undergoing the same operation and reaction (Casciotti et al., 2002 2002 ). However, the measurement precision of δ 18 O was low and the contraction ratios of δ 18 O were high under non-sealed culture conditions. Different amplitudes of δ 18 O contractions were observed under sealed-culture conditions with different gas-liquid ratios in culture flasks. That is, DO concentrations had a substantial influence on the growth of denitrifying bacteria and the conversion of nitrate to N 2 O, and this may be the most significant contributor to δ 18 O contraction. During denitrification, oxygen, nitrite, and nitrate are electron acceptors for nitrate conversion to N 2 O in P. aureofaciens (Hu et al., 2018 2018 ). If oxygen is sufficient, the bacteria can grow quickly through aerobic respiration. When oxygen drops below a certain concentration, the bacteria will switch to nitrate respiration and gain electrons from the respiratory chain (Galloway et al., 2004 2004 ; Huang and Xin, 2009 2009 ). McIlvin and Casciotti ( 2011 2011 ) found that the cell density of a pure culture in an anaerobic environment was only 5% of that in an aerobic environment. It seems that the throughput of the denitrifier method can be improved using non-sealed cultures. However, higher DO concentrations may result in more disturbances to the δ 18 O measurement of RMs so that the differences among the δ 18 O values of RMs become indistinguishable (Fig. 2 2 ). When DO concentrations are high, nitrite reductase activity is inhibited, and the carbon source is broken down quickly to produce cells. This results in a lack of sufficient electron donors in subsequent denitrification and the accumulation of intermediate products. Therefore, appropriate DO concentrations in pure culture are essential for microbial growth and influence the completeness of nitrate conversion to N 2 O. DO concentrations in the medium depend on the dissolution rate of oxygen, gas-liquid contact time and area, gas volume, oxygen partial pressure, and the nature of the medium (Tiedje, 1988 1988 ). Thus, DO concentrations were controlled by the gas-liquid ratio of culture flasks and the shaking speed under sealed conditions. Two shaking speeds (60 and 120 r/min) were tested to explore the appropriate DO concentration under a gas-liquid ratio of 3:10 (the area of gas-liquid was about 3.7 cm 2 ). Bacterial film and flocculent precipitation were observed at a shaking speed of 60 r/min, which made it difficult to centrifuge and blend. The measured isotope ratios varied significantly, and coenobium was broken up by vortex before centrifugation in the case of 60 r/min. By contrast, the 120 r/min shaking speed allowed the growth of bacterial turbidity, but no floc, which resulted in better measurement precision of the RMs (Table 1 1 ). Generally, denitrification is complete when the DO concentration is less than 0.2 mg/L (Downes, 1988 1988 ). Most aerobic denitrifying bacteria can tolerate DO concentrations below 3 mg/L (Zhou et al., 2007 2007 ). However, nitrate may be still selected as an electron acceptor in the process of denitrification under a wider range of DO concentrations (2.3 to 11.3 mg/L) for some aerobic denitrifying bacteria (Wang et al., 2007 2007 ). Our results indicated that δ 18 O estimates of nitrate were more precise when the DO concentrations were controlled by a gas-liquid ratio of 3:10 and a shaking speed of 120 r/min. The concentrations of DO (mg/L) were 1.9 on the second, 2.6 on the fifth, and 2.2 on the seventh day of pure culture. However, the DO concentrations were more than 3 mg/L under other oxygen conditions (i.e. non-sealed culture or gas-liquid ratios of 2:5 and 3:5), which resulted in poor performance for isotopic measurement, especially for the oxygen isotope. Accordingly, the threshold values of DO concentrations for P. aureofaciens should be below 3 mg/L. The reason why isotopic measurement was poor at DO concentrations above 3 mg/L might be that excessive DO inhibits the activity of nitrite reductase and causes the accumulation of intermediate products (Fig. 3 3 ) (Bu et al., 2015 2015 ). Kumar and Lin ( 2010 2010 ) confirmed that the reductase in the denitrification process was sensitive to oxygen, and its activity was affected by DO concentrations. A high DO concentration may promote oxygen atom exchange between intermediate products and distilled water used for the preparation of medium in the laboratory. δ 18 O values of distilled water of less than 0‰ have been reported (Koehler et al., 1991 1991 ). Thus, it can be inferred that the δ 18 O values of RMs, especially those of N3, which were lower than the true value, may be the result of oxygen atoms exchanging with distilled water. The degree of exchange was closely related to the DO concentration, and high DO concentrations may generate dilution of oxygen isotopes in intermediate products. Therefore, the effect of intermediates on the conversion of nitrate in samples to N 2 O was significantly greater in high than in low DO concentrations. Eventually, the measured values of δ 18 O in different RMs were indistinguishable due to the high DO concentration and high degree of oxygen atom exchange with water. Precision analysis 2 Fig. 4 4 shows the results of linear regression analysis of four RMs (N3, USGS34, USGS32:USGS34, and USGS32:2USGS34) ( R 2 =1, P < 0.001). Verified three times, the DO concentrations (controlled by a gas-liquid ratio of 3:10 and a shaking speed of 120 r/min) and the nitrate sample sizes (0.2 to 0.4 μmol) were effective and feasible. The slight δ 18 O contraction can be corrected by the standard curve. U2:U4 and U2:2U4 solutions were prepared by mixing USGS32 (U2) and USGS34 (U4) in the proportions of 1:1 and 1:2, respectively. n =3 Eight rainwater samples were collected from a watershed in northwestern Zhejiang Province, China by a rainwater collector in Jan. 2017. The δ 15 N-NO 3 − and δ 18 O-NO 3 − values were determined with 0.2 μmol NO 3 − . The measured results are showed in Fig. 5 5 . The δ 15 N-NO 3 − values ranged from −0.04‰ to 4.02‰ with an average SD of 0.16‰, and the δ 18 O-NO 3 − values ranged from 36.54‰ to 81.03‰ with an average SD of 0.25‰ ( n =3). In general, δ 15 N-NO 3 − derived from precipitation ranges from −10‰ to 8‰ (Rogers et al., 2012 2012 ), and δ 18 O-NO 3 − from 25‰ to 75‰ (Yang et al., 2013 2013 ). Compared with the δ 18 O precision (0.5‰ to 2.0‰) of the water samples reported in previous studies, we obtained a slightly higher precision in this study. The bars represent the standard deviations Conclusions 1 In general, our study revealed a strong relationship between δ 18 O measurements and DO concentrations or nitrate sample sizes. DO concentration in pure culture affected the subsequent conversion of nitrate to N 2 O in denitrifying culture, because it promoted the accumulation of intermediate products and oxygen exchange with water. Ultimately, DO concentrations affected the measurement precision and accuracy of δ 15 N and δ 18 O, especially δ 18 O. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1800197 Project supported by the National Key R&D Program of China (No. 2016YFD0200106) and the National Natural Science Foundation of China (No. 41571450) 2018-03-31 2018-07-22 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 20