郭茹(1997—),女,山西运城人,硕士研究生,主要从事土壤氮转化研究。E-mail:
This paper aims to optimize the culture and reaction conditions of
There was no significant difference in the 15N isotopic determination of
The optimized method can accurately determine the 15N abundance of
土壤氮转化过程是生物地球化学循环的重要环节。土壤中的氮可分为有机态氮和无机态氮,不同形态的氮在生物和化学作用下可相互转化。在无机态氮中
反硝化细菌法采用缺乏N2O还原酶活性的兼性反硝化细菌,将
本研究在Böhlke等[
本文所用菌种为
本实验使用的
为保证不同批次菌体的稳定性、减少染菌风险、节约菌体前期生长培养时间,采用Weigand等[
(1)接种方式的优化。对反硝化细菌
(2)培养方式的优化。在不同批次间设置微氧摇培和好氧摇培两种情况。微氧摇培参考Böhlke等[
(3)反硝化细菌浓度优化。为考察反硝化细菌浓度对
(1)气体吹扫优化。反硝化细菌法需使用惰性气体吹扫创造厌氧环境,去除残留空白(
(2)气体保存优化。设置转移气体和不转移气体两种气体保存方式,各设置3个重复。转移气体处理需使用带锁帽的注射器从22.5 mL培养瓶顶空抽取约12~14 mL气体,转移至预先抽真空的含1~2片NaOH固体的22.5 mL顶空瓶中,补入14 mL高纯He保持正压状态。不转移气体处理是直接在含有样品和菌液的培养瓶中,使用带双套针的进样装置连接培养瓶,需当天在质谱仪上完成测样。
(1)不同浓度KCl对
(2)不同土地利用类型土壤中
实验中所使用的同位素标样(
本文使用的同位素标准样品
Bulk isotopic data for materials used in this study
样品名称1) |
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注:1)Lab-1是本实验室配制的NaNO2标准样品,RSIL是购自Reston Stable Isotope Laboratory的NaNO2标准样品,KNO2-A、KNO2-B、KNO2-C是本实验室配制的三个不同 |
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Lab-1 | –13.58 | / |
RSIL-N7373 | –79.6 | +4.5 |
RSIL-N10219 | +2.8 | +88.5 |
RSIL-N23 | +3.7 | +11.4 |
KNO2-A | +112.38 | / |
KNO2-B | +165.00 | / |
KNO2-C | +422.17 | / |
本文中测定得到的
分别使用单菌落、种子液两种接种方式培养
培养条件的优化对测定样品的峰面积对比(a)及好氧摇培
Optimization of culture conditions on the comparison of peak areas of measured samples(a)and growth curves of aerobic shake culture of
微氧摇培和好氧摇培两种方式对Lab-1标样的
反硝化细菌法测定
反硝化菌法测定
反硝化反应时菌体浓度的高低可能影响测定结果。分别对不同OD600值菌体进行两批次的实验结果显示(
菌体OD600值对
Influence of OD600 value of bacteria on the
20 μmol·L–1 |
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注:KNO2-A、KNO2-B、KNO2-C均为本实验室配制的标记丰度溶液, |
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菌体OD600 |
KNO2-A | KNO2-B | KNO2-C | ||||||||
SD | SD | SD | |||||||||
0.42 | 107.67 | 0.51 | 5 | 158.00 | 0.96 | 5 | 409.26 | 2.32 | 5 | ||
0.67 | 107.51 | 0.57 | 5 | 157.83 | 0.76 | 5 | 407.03 | 2.77 | 5 | ||
0.78 | 107.55 | 0.29 | 5 | 158.00 | 0.28 | 5 | / | / | / | ||
1.18 | 106.34 | 0.15 | 4 | 156.18 | 0.33 | 4 | / | / | / | ||
20 μmol·L–1 |
|||||||||||
菌体OD600 |
RSIL-N7373 | RSIL-N10219 | RSIL-N23 | ||||||||
SD | SD | SD | |||||||||
0.29 | –74.26 | 0.37 | 3 | 2.37 | 0.04 | 3 | 3.72 | 0.16 | 3 | ||
0.32 | –74.78 | 0.14 | 3 | 2.21 | 0.04 | 3 | 3.65 | 0.03 | 3 | ||
0.58 | –74.36 | 0.09 | 3 | 2.23 | 0.12 | 3 | 3.59 | 0.00 | 3 | ||
0.60 | –74.42 | 0.56 | 3 | 1.10 | 0.10 | 3 | 3.23 | 0.10 | 3 | ||
0.70 | –74.26 | 0.11 | 3 | 2.04 | 0.13 | 3 | 3.34 | 0.12 | 3 | ||
0.88 | –75.93 | 0.52 | 3 | 0.94 | 0.06 | 3 | 2.99 | 0.04 | 3 | ||
2.10 | –42.85 | 3.13 | 3 | –5.09 | 0.39 | 3 | –4.51 | 0.16 | 3 |
已有研究表明,反硝化细菌法测定
土壤浸提液中含有大量K+、Cl–,可能影响反硝化菌体活力,分别测定了0.1 mol·L–1、0.5 mol·L–1、1 mol·L–1、2 mol·L–1 KCl配制的Lab-1标样的
不同浓度KCl(0.1,0.5,1,2 mol·L–1)对样品
Effect of different concentrations of KCl(0.1, 0.5, 1, 2 mol·L–1)on the
使用上述优化后的反硝化细菌法反应体系,测定不同pH的农田、水稻、森林土壤中
不同土地利用类型土壤
Results of 15N abundance measurements for different types of land use soil
土地利用类型Land use types | pH | ||
反硝化细菌法Denitrifier method | 化学转化法Chemical method | ||
注:人为添加了20 nmol的 |
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旱地(四川盐亭) |
7.96 | –13.46±0.28 | –13.81±0.21 |
水田(广西桂林) |
7.28 | –14.06±0.04 | –14.81±0.23 |
水田(吉林长春) |
6.23 | –13.84±0.08 | –14.11±0.38 |
水田(安徽安庆) |
5.23 | –14.26±0.05 | –13.44±0.57 |
林地(江西鹰潭) |
4.40 | –13.68±0.17 | –12.78±0.41 |
本文针对
Müller C, Clough T J. Advances in understanding nitrogen flows and transformations: Gaps and research pathways[J]. The Journal of Agricultural Science, 2014, 152(S1): 34-44.
Russow R, Stange C F, Neue H U. Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: Results from 15N tracer experiments[J]. Soil Biology & Biochemistry, 2009, 41(4): 785-795.
Rütting T, Müller C. Process-specific analysis of nitrite dynamics in a permanent grassland soil by using a Monte Carlo sampling technique[J]. European Journal of Soil Science, 2008, 59(2): 208-215.
Maharjan B, Venterea R T. Nitrite intensity explains N management effects on N2O emissions in maize[J]. Soil Biology & Biochemistry, 2013, 66: 229-238.
Su H, Cheng Y F, Oswald R, et al. Soil nitrite as a source of atmospheric HONO and OH radicals[J]. Science, 2011, 333(6049): 1616-1618.
Butterbach-Bahl K, Baggs E M, Dannenmann M, et al. Nitrous oxide emissions from soils: How well do we understand the processes and their controls?[J]. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 2013, 368(1621): 20130122.
Yang F, Dai S Y, Zhang J B, et al. Nitrite transformations under acidic conditions in temperate and subtropical forest ecosystems[J]. Geoderma, 2018, 317: 47-55.
Dai S Y, Wen T, Cai Z C, et al. Dynamics of nitrite in acidic soil during extraction with potassium chloride studied using 15N tracing[J]. Rapid Communications in Mass Spectrometry, 2020, 34(9): e8746.
Cao Y C, Zhong M, Gong H, et al. Determining 15N abundance in ammonium, nitrate and nitrite in soil by measuring nitrous oxide produced[J]. Acta Pedologica Sinica, 2013, 50(1): 113-119.
曹亚澄, 钟明, 龚华, 等. N2O产生法测定土壤无机态氮15N丰度[J]. 土壤学报, 2013, 50(1): 113-119.
Stevens R J, Laughlin R J. Determining nitrogen-15 in nitrite or nitrate by producing nitrous oxide[J]. Soil Science Society of America Journal, 1994, 58(4): 1108-1116.
Silva S R, Kendall C, Wilkison D H, et al. A new method for collection of nitrate from fresh water and the analysis of nitrogen and oxygen isotope ratios[J]. Journal of Hydrology, 2000, 228(1/2): 22-36.
McIlvin M R, Altabet M A. Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater[J]. Analytical Chemistry, 2005, 77(17): 5589-5595.
Isobe K, Koba K, Suwa Y, et al. Nitrite transformations in an N-saturated forest soil[J]. Soil Biology & Biochemistry, 2012, 52: 61-63.
Lachouani P, Frank A H, Wanek W. A suite of sensitive chemical methods to determine the δ15N of ammonium, nitrate and total dissolved N in soil extracts[J]. Rapid Communications in Mass Spectrometry, 2010, 24(24): 3615-3623.
Tu Y, Fang Y T, Liu D W, et al. Modifications to the azide method for nitrate isotope analysis[J]. Rapid Communications in Mass Spectrometry, 2016, 30(10): 1213-1222.
Sigman D M, Casciotti K L, Andreani M, et al. A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater[J]. Analytical Chemistry, 2001, 73(17): 4145-4153.
Casciotti K L, Böhlke J K, McIlvin M R, et al. Oxygen isotopes in nitrite: Analysis, calibration, and equilibration[J]. Analytical Chemistry, 2007, 79(6): 2427-2436.
Granger J, Sigman D M, Prokopenko M G, et al. A method for nitrite removal in nitrate N and O isotope analyses[J]. Limnology and Oceanography: Methods, 2006, 4(7): 205-212.
Böhlke J K, Smith R L, Hannon J E. Isotopic analysis of N and O in nitrite and nitrate by sequential selective bacterial reduction to N2O[J]. Analytical Chemistry, 2007, 79(15): 5888-5895.
Finkmann W, Altendorf K, Stackebrandt E, et al. Characterization of N2O-producing
Nielsen M, Larsen L H, Jetten M S M, et al. Bacterium-based NO2- biosensor for environmental applications[J]. Applied and Environmental Microbiology, 2004, 70(11): 6551-6558.
Dai S Y, Wen T, Zhang J B, et al. Improving extraction method in determining NO2--N in acidic soil[J]. Soils, 2018, 50(2): 341-346.
戴沈艳, 温腾, 张金波, 等. 酸性土壤中亚硝态氮提取方法的改进[J]. 土壤, 2018, 50(2): 341-346.
Rock L, Ellert B H. Nitrogen-15 and oxygen-18 natural abundance of potassium chloride extractable soil nitrate using the denitrifier method[J]. Soil Science Society of America Journal, 2007, 71(2): 355-361.
Bell M D, Sickman J O. Correcting for background nitrate contamination in KCl-extracted samples during isotopic analysis of oxygen and nitrogen by the denitrifier method[J]. Rapid Communications in Mass Spectrometry, 2014, 28(5): 520-526.
Mørkved P T, Dörsch P, Søvik A K, et al. Simplified preparation for the δ15N-analysis in soil NO3- by the denitrifier method[J]. Soil Biology & Biochemistry, 2007, 39(8): 1907-1915.
Zhu J, Yu L, Bakken L R, et al. Controlled induction of denitrification in Pseudomonas aureofaciens: A simplified denitrifier method for dual isotope analysis in NO3-[J]. Science of the Total Environment, 2018, 633: 1370-1378.
Weigand M A, Foriel J, Barnett B, et al. Updates to instrumentation and protocols for isotopic analysis of nitrate by the denitrifier method[J]. Rapid Communications in Mass Spectrometry, 2016, 30(12): 1365-1383.
McIlvin M R, Casciotti K L. Technical updates to the bacterial method for nitrate isotopic analyses[J]. Analytical Chemistry, 2011, 83(5): 1850-1856.
Stock P, Roder S, Burghardt D. Further optimisation of the denitrifier method for the rapid 15N and 18O analysis of nitrate in natural water samples[J]. Rapid Communications in Mass Spectrometry, 2021, 35(1): e8931.
Casciotti K L, Sigman D M, Hastings M G, et al. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method[J]. Analytical Chemistry, 2002, 74(19): 4905-4912.
Well R, Flessa H, Lu X, et al. Isotopologue ratios of N2O emitted from microcosms with NH4+ fertilized arable soils under conditions favoring nitrification[J]. Soil Biology & Biochemistry, 2008, 40(9): 2416-2426.