Controlled induction of denitrification in Pseudomonas aureofaciens: A simplified denitrifier method for dual isotope analysis in NO3−
Controlled induction of denitrification in Pseudomonas aureofaciens: A simplified denitrifier method for dual isotope analysis in NO3−
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金黄色假单胞菌反硝化的受控诱导：用于 NO3 中双同位素分析的简化反硝化器方法
DOI：
10.1016/j.scitotenv.2018.03.236
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发表时间：
2018
影响因子：
9.8
通讯作者：
Peter Dörsch
中科院分区：
文献类型：
--
作者：
Jing Zhu;Longfei Yu;Lars R. Bakken;Pål Tore Mørkved;Jan Mulder;Peter Dörsch
硝酸盐（NO3−）的δ15N和δ18O双同位素特征对限制生物圈中氮转化过程具有重要意义。通过缺乏N2O还原酶基因编码的反硝化菌生物转化为氧化亚氮（N2O）已成为从自然样品中分离NO3 -用于15n和18o分析的首选方法（“反硝化菌法”）。该方法的成功取决于培养物快速和定量地将NO3−转化为N2O的能力，同时尽可能少地受到非样品NO3−的污染或水18o的干扰。我们研究了假单胞菌缺氧转化。金黄色葡萄球菌在复杂培养基中氧化生长。当让培养物从充足的o2和mM NO3−开始变为缺氧时，一氧化氮积累到有毒水平，损害反硝化作用。在最初O2availability和μM NO3−较低的情况下，有效转化为N2O取决于mM铵的存在。在较高的细胞密度下，转化效率降低，表明诱导平衡反硝化的能力依赖于生长。我们的结果表明p。在μM NO3−存在的情况下，金黄色葡萄球菌可以成功诱导反硝化，使外源NO3−的缺氧预培养失效。我们设计了一种简化的反硝化方法，该方法快速可靠，不需要浓缩细胞或去除外来的NO3−或N2O。培养基中的背景NO3−可以通过p的缺氧预孵育以n2的形式去除。denitrificans。δ15N的自然丰度重复性为0.1 ~ 0.3‰，δ18O的自然丰度重复性为0.2 ~ 0.7‰。我们成功地测试了各种样品基质的方法，包括低pH （pH = 4）和高盐度（0.25 - 1 M KCl）。在高度15n标记的NH4+或氨基酸存在的情况下分析NO3 -同位素，没有发现交叉污染。我们得出结论，简化的反硝化方法非常适合于分析广泛的自然样品中的双NO3−同位素。
Dual isotope signatures (δ15N and δ18O) of nitrate (NO3−) are invaluable to constrain nitrogen transformation processes in the biosphere. Biological conversion to nitrous oxide (N2O) by denitrifiers lacking the gene coding for N2O reductase has become the method of choice for isolating NO3−from natural samples for15N and18O analysis (“denitrifier method”). The success of the method depends on the ability of the culture to rapidly and quantitatively convert NO3−to N2O with as little as possible contamination from non-sample NO3−or interference with water18O. We studied oxic-anoxic transition inPseudomonaschlororaphisss.aureofaciensgrown oxically in complex medium. When letting the culture turn anoxic starting with ample O2and mM NO3−, nitric oxide accumulated to toxic levels, impairing denitrification. With initially low O2availability and μM NO3−, efficient conversion to N2O depended on the presence of mM ammonium. At higher cell densities, conversion efficiencies decreased, suggesting that the ability to induce balanced denitrification is growth dependent. Our results show thatP. aureofacienscan induce denitrification successfully in the presence of μM NO3−, making anoxic pre-culturing with extraneous NO3−obsolete. We devise a simplified denitrifier method, which is fast and robust and does not require concentration of cells or removal of extraneous NO3−or N2O. Background NO3−in the medium can be removed as N2by anoxic pre-incubation withP. denitrificans. Repeatability at natural abundance ranged from 0.1–0.3‰ for δ15N and 0.2–0.7‰ for δ18O. We successfully tested the method for various sample matrices including low pH (pH = 4) and high salinity (0.25 - 1 M KCl). Analyzing NO3−isotopes in the presence of highly15N-labelled NH4+or amino acids showed no cross-contamination. We conclude that the simplified denitrifier method is well suited for analyzing dual NO3−isotopes in a wide range of natural samples.