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基于14NH 4 + /4+ 15NH 4= 通过顺序转换为 N2O 的分析，测量异化硝酸盐降低至铵的潜在率

Published: October 07, 2020
doi:
10.3791/59562
, , , , , ,
1Department of Biological Sciences, Faculty of Science and Engineering,Chuo University, 2Faculty & Graduate School of Urban Environmental Sciences,Tokyo Metropolitan University, 3Center for Ecological Research,Kyoto University, 4Department of Chemistry, Faculty of Science,Toho University, 5Graduate School of Integrated Arts and Sciences,Hiroshima University

Summary

详细介绍了基于 14 NH 4+ / 15NH444+分析确定潜在 DNRA 速率的一系列方法。NH4+通过几个步骤转换为 N2O，并使用四极气相色谱法（质谱法）进行分析。

Abstract

由于全球氮负荷在工业化后急剧增加，了解硝酸盐（NO3+）的命运的重要性不断增加，硝酸盐是从陆地向水生生态系统转移的主要N种。异硝酸盐减少至铵（DNRA）和脱硝是使用NO 3的微生物过程，用于呼吸。与否认相比，对DNRA活动的定量测定只进行了有限的程度。这导致对 DNRA 在 NO3中的重要性认识不足 –转换和此过程的调节因素。本文的目的是提供一个详细的程序，用于测量环境样品中潜在的DNRA速率。简言之，潜在的DNRA率可以从15N标铵（15NH154+）+的积累率（15NO3=添加孵化）中计算。本文所述的14NH4+和15NH4°浓度的确定由以下步骤组成。首先，提取样品中的NH4+，+并捕获在酸化玻璃过滤器作为铵盐。其次，通过硫酸盐氧化，将被困的铵洗洗并氧化为NO 3。第三，NO3=通过 N2O 还原酶缺乏除尼器转换为 N2O。最后，使用先前开发的四极气相色谱法-质谱系统对转换后的N2O进行了分析。我们将这种方法应用于盐沼沉积物，并计算了其潜在的DNRA速率，表明与前面描述的方法相比，建议的程序允许简单、更快速地确定。

Introduction

氮肥的人工合成及其广泛应用极大地干扰了全球氮循环。据估计，自工业化前1次以来，活性氮从陆地系统向沿海系统的转移增加了一倍。应用于给定田地的肥料的很大一部分被从土壤中冲走到河流或地下水中，主要为 NO3+ 2。这可能会导致环境问题，如饮用水污染，富营养化，和缺氧的形成。NO3=在水环境中，通过生物同化和各种微生物异化过程，从生态系统中去除或保留。脱硝和麻醉剂是NO3+的主要微生物去除过程。硝化是微生物还原NO3=气态N产物（NO、N2O和2N2）加上电子供体（如有机物质）的氧化，从而降低了上述问题的风险。Anammox 还从 NO 2 + 和NH4+ 生成 N2 ;−因此，它将无机N从生态系统中删除。相反，DNRA 致力于在生态系统中保留 N;人们普遍认为，DNRA主要由发酵细菌或化学营养细菌进行，它们可减少异化NO 3–生物利用和移动性较少的NH4++。

关于DNRA的研究主要在海洋或河口生态系统中进行，如海洋或河口沉积物和水、盐或咸沼泽土壤以及红树林土壤。,沿海或海洋生态系统作为从陆地生态系统中去除NO,− 3+−的蓄水池非常重要，在以前的研究中，DNRA已证明在NO 3 + 清除 （0-99%）,3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18,46,7,8,9,10,11,12,15,16,17的范围内13,14做出了贡献18。35此外，DNRA的存在已证明在广泛的环境，包括淡水环境19，稻田土壤20，森林土壤21。虽然这些研究表明，DNRA与NO 3+去除的脱硝具有潜在可比性，但与测量脱硝量相比，测量DNRA活性的研究仍然非常有限。

DNRA 速率通过分析或数值模型与数据分析相结合，使用15种 N 标标技术进行评估。一个用于计算 DNRA 速率的分析解决方案是基于在添加 15NO3=作为示踪剂后 NH4+池的 15N 浓缩量的增加。15N标记的NO3=被添加到样品中孵育，然后可以从NH 4+的浓度和同位素比变化中计算出在一定时期之前和之后。本文详细介绍了计算DNRA速率所需的NH4++浓度和同位素比的量化方法。基本上，这里报告的方法是结合了几个以前报告的技术22，23，24，25，26，修改添加到一些程序。22,23,24,25,26该方法由一系列五个组分程序组成：（1） 利用稳定同位素示踪剂的修正孵育环境−样品，15NO3+，（2） 使用”扩散程序”提取和恢复 NH4+，并修改，（3） 在样品中对 NH4+进行吸气氧化， 由土著 NH4+和 15NH4+从 15No3=通过 DNRA 活性派生为 No 3+和15No3+， （4） 随后的3 号微生物转化和 15NO3= 到N2O 异体通过修改的除色器方法， 和 （5） 使用气相色谱法 （GC/MS） 定量 N2O 异体体。3在以下一节中，首先对程序 （2） 和 （4） 的准备进行了描述，然后详细描述了所有五个组成部分过程。

Protocol

1. 准备 PTFE 信封，用于定量捕获气态 NH3 将一块 60 毫米的聚四氟乙烯 （PTFE） 胶带（25 毫米宽）放在一小块铝箔上（约 300 mm x 450 mm 大小，用乙醇擦拭）。 在 450°C 下将玻璃纤维过滤器（直径为 10 mm，孔径为 2.7 μm）灰，在消声炉中为 4 小时。将玻璃纤维过滤器放在胶带长轴的中点上方一点（图 1a）。 在 GF/D 过滤器的中心点 20 μL 的 0.9 摩尔/L H<…

Representative Results

本文提出的代表性结果来自 盐沼沉积物的15个N-跟踪实验。采样盐沼是在2011年日本东部大地震后在日本宫城县Kesen-numa市的Moune地区新建的。2017年9月，在部下和部间区的两个地点收集了地表沉积物（0~3厘米）。首先，在收集后，沉积物立即通过4毫米网状，清除植物根、贝壳碎片和碎石，然后均质化。样品储存在4°C，直到DNRA分析进行。 <p class="jove_content" fo:keep-to…

Discussion

DNRA分析NH4+的浓度和同位素比用几种方法进行量化。NH4+的浓度和同位素比通常单独测量。NH4+浓度通常使用色度测量方法测量，包括自动分析仪4、,10、15、16、17。同位素比测量具有很大的变化，取决于其NH4+转换</su…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢田中真纪子帮助数据收集和开发协议。样本收集由JSPS KAKENHI赠款编号17K15286支持。

Materials

15N-KNO3 SHOKO SCIENCE N15-0197
15N-NH4Cl SHOKO SCIENCE N15-0034
20 mL PP bottle SANPLATEC 61-3210-18 Wide-mouth
Aluminum cap Maruemu 1307-13 No. 20, with hole
Boric acid Wako 021-02195
Centrifuge HITACHI Himac CR21G II
Deoxygenized Gas Pressure & Replace Injector SANSIN INDUSTRIAL IP-12
Disposable cellulose acetate membrane filter ADVANTEC 25CS020AS Pore size 0.22 µm, 25 mm in diameter
Disposable syringe Termo SS-10SZ 10 mL
Disposable syringe Termo SS-01T 1 mL
Dulbecco’s Phosphate Buffered Saline (-) NISSUI PHARMACEUTICAL 5913
Gastight syringe VICI Valco Instruments 4075-15010 Series A-2, 100 µL
GC/MS shimadzu GCMS-QP2010ultra
GF/D Whatman 1823-010 10 mm in diameter
Glass vial Maruemu 0501-06 20 mL
Gray butyl rubber stopper Maruemu 1306-03 No.20-S
H2SO4 Wako 192-04696 Guaranteed Reagent
K2S2O8 Wako 169-11891 Nitrogen and Phosphorus analysis grade
KCl Wako 163-03545 Guaranteed Reagent
KNO3 Wako 160-04035 Guaranteed Reagent
NaOH Wako 191-08625 Nitrogen compounds analysis grade
NH4Cl Wako 017-02995 Guaranteed Reagent
Plastic centrifuge tube ASONE 1-3500-22 50 mL, VIO-50BN
Pseudomonas chlororaphis subsp. aureofaciens American Type Culture Collection (ATCC) ATCC 13985 Freeze-dried, the type strain of Pseudomonas aureofaciens
PTFE sealing tape Sigma-Aldrich Z221880 25 mm in width
Reciprocating shaker TAITEC 0000207-000 NR-10
Screw-cap test tube IWAKI 84-0252 11 mL
PTFE-lined cap for test tube IWAKI 84-0262
Tryptic Soy Broth Difco Laboratories 211825
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Tags

DNRANitrate ReductionAmmoniumN15 AnalysisN2OGas Chromatography-mass SpectrometryCoastal EcosystemsAquatic EnvironmentsDenitrificationAnammox

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Kuroiwa, M., Fukushima, K., Hashimoto, K., Senga, Y., Sato, T., Katsuyama, C., Suwa, Y. Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O. J. Vis. Exp. (164), e59562, doi:10.3791/59562 (2020).
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