CN111518863A - 一种测定沉积物全程硝化反应速率及对氨氮去除率的方法 - Google Patents
一种测定沉积物全程硝化反应速率及对氨氮去除率的方法 Download PDFInfo
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Abstract
本发明公开了一种测定水体沉积物全程硝化反应速率及其对氨氮去除贡献率的方法,属湿地氮污染控制领域。其原理是利用氯酸盐对亚硝酸盐氧化酶和辛炔对氨氧化细菌和古菌的选择性抑制,测定沉积物中全程硝化微生物的反应速率,并计算其对氨氮去除的贡献率。主要步骤包括:(1)待测沉积物的抑制剂处理及培养:①不添加,②添加氯酸钾,③添加氯酸钾和辛炔;(2)测培养过程中沉积物NO3 ‑和NO2 ‑浓度;(3)与培养时间线性回归得全程硝化反应速率;(4)计算全程硝化对氨氧化去除的贡献率。该方法能模拟原位温度、营养元素、含水率等,可精确、高效地测定自然界多种水体沉积物中全程硝化反应速率及其对氨氮去除率的研究,推动全程硝化研究的发展。
Description
技术领域
本发明涉及湿地氮污染控制领域,新型氮循环微生物全程硝化细菌规避了硝化过程限速步骤-氨氧化过程,可实现水生态系统中的氨氮污染物的快速去除,并且避免有毒中间产物亚硝酸盐的产生;同时由于其较传统两步法而言反应电势更低,因此更加广泛的作用于自然环境中氨氮去除,为水环境水生态中氨氮治理提供新思路。
背景技术
氮(N)循环主要由微生物驱动,微生物通过一系列氧化还原反应转化为不同的氮化合物。以不同氧化还原梯度为特征的两相之间的界面往往是生物地球化学N循环的热区。硝化作用是氮循环的限速步骤,是微生物将氨连续氧化成硝酸盐的生物学过程,是生物地球化学氮循环和生物废水处理工艺中的关键过程,也应用在农业的许多方面而发挥重要作用。自19世纪90年代发现以来,一直认为硝化作用由两类不同的化能自养型细菌:氨氧化微生物(氨氧化细菌(ammonium-oxidizing bacteria,AOB)和氨氧化古菌(ammonium-oxidizing Archaea,AOA))和亚硝酸盐氧化细菌(nitrite-oxidizing bacteria,NOB))完成的两步氮循环过程。尽管研究表明氨氧化古菌(ammonium-oxidizing archaea,AOA)在土壤和水生系统的硝化过程中发挥着重要作用,但这并没有改变对硝化作用的传统认识,即氨是由微生物经两步氮循环过程最终被氧化为硝酸盐。随后,厌氧氨氧化(anammox)的发现表明在缺氧和厌氧条件下氨氧化过程的真实存在,其与传统的氨氧化过程完全不同。新的氮循环的过程的不断发现表明,我们对总氮循环的认知还不进完整。Costa等认为,从微生物能量代谢的角度分析,理论上存在完全硝化的细菌,但并没能获得直接的证据。最近,有研究表明,Nitrospira属中的微生物可以独立完成氨氧化成硝酸盐的氮代谢过程,并将这类微生物定义为全程硝化微生物。这一过程的发现使我们对硝化作用有了一个全新的认识和理解。近年来,在人造水生和耕作土壤生态系统中已广泛检测到全程硝化细菌,但全程硝化细菌对自然生态系统中氨氧化过程和总氮循环的贡献仍然不清楚。
发明内容
本发明的目的就是为了解决上述问题,提出了一种双抑制剂法测定沉积物中全程硝化微生物反应速率并计算其对氨氧化贡献率的方法,即利用氯酸盐对亚硝酸盐氧化酶和辛炔对氨氧化细菌和氨氧化古菌的选择性抑制作用,通过在沉积物中添加抑制剂处理培养后,产物和的变化,测定水/沉积物体系全程硝化反应速率,并通过进一步计算获得全程硝化对氨氮去除贡献率。
本发明通过以下样品处理和数据分析方案来实现:
(1)双抑制剂培养
称取等质量的新鲜沉积物样品到若干60毫升血清瓶中,加盖胶塞和铝盖,密封避光培养。随机挑选血清瓶做三种不同抑制剂处理。处理1-空白组,添加1.5毫升无菌水到每一个血清瓶中;处理2-氯酸盐抑制剂处理组,添加1.5ml 0.13mol/L的氯酸钾到血清瓶中;处理3-氯酸盐+辛炔双抑制剂处理组,添加1.5ml 0.13mol/L的氯酸钾和2kPa的辛炔到血清瓶。每个处理均做3组平行。所有血清瓶放置于恒温摇床在转速150rpm和原位温度进行培养,于培养开始后的第0天、1天、2天和4天分别陆续停止培养。添加20ml 2mol/L的KCl到相应培养时间的血清瓶中,于150rpm摇床震荡15min,将沉积物中的亚硝酸盐和硝酸盐提取到液相中,再经过离心过滤处理,收集上清液待测。
(2)产物分析
全程硝化速率通过(处理1)和(处理2)的累积速率差来计算得出,其中,的积累主要因为亚硝酸氧化过程受抑制;关于AOA和AOB速率,主要是基于处理2中由AOA和AOB而产生,而没有NOB消耗;处理3中AOB和NOB均被抑制,NO2-仅由AOA反应得出。AOA速率是通过计算(处理2)的累积速率得出;AOA加AOB速率是通过在处理2和处理3中的积累速率差来得出;AOB速率同时AOA加AOB速率减去AOA速率得出。
公式1和公式2中,指浓度,指浓度,指亚硝酸盐中氮元素的浓度,指硝酸盐中氮元素的浓度,VKCl指浸提沉积物所添加的KCl溶液体积,指在处理1和处理2的血清瓶沉积物中添加的无菌水或者KClO3溶液体积,Msediment指各血清瓶中新鲜沉积物的质量。在本发明中,VKCl优选设置为20mL,优选设置为1.5mL,Msoil优选设置为5g。
(3)回归计算
根据公式4和公式6,可以得出AOB的反应速率,如公式7。
根据公式6和公式9,可以得出NOB的反应速率,如公式10。
根据公式9和公式12,可以得出comammox的反应速率,如公式13。
Claims (11)
1.一种测定水体沉积物全程硝化反应速率及其对氨氮去除率的方法,包括步骤如下:
(1)双抑制剂培养:将新鲜沉积物样品放置于血清瓶中,加盖胶塞和铝盖,密封避光,对血清瓶分别做不同抑制剂处理,并于原位温度和DO条件培养;
(2)产物分析:提取并测定不同培养时间下,沉积物样品中产物NO3 -和NO2 -的浓度,并计算相应的积累速率;
(3)回归计算:通过三个不同处理之间产物NO3 -和NO2 -的累积速率变化,计算沉积物中全程硝化反应速率及对氨氮去除贡献率。
2.根据权利要求1所述,本方法优选60毫升血清瓶用于双抑制剂培养。
3.根据权利要求1所述,本方法优选添加1.5毫升无菌水到每一个血清瓶,作为处理1-空白组。
4.根据权利要求1所述,本方法优选添加1.5ml 0.13mol/L的氯酸钾到血清瓶中,作为处理2-氯酸盐抑制剂处理组。
5.根据权利要求1所述,本方法优选添加1.5ml 0.13mol/L的氯酸钾和2k Pa的辛炔到血清瓶,作为处理3-氯酸盐+辛炔双抑制剂处理组。
6.根据权利要求1所述,本方法特征要求每个处理均做3组平行。
7.根据权利要求1所述,本方法优选血清瓶放置于恒温摇床在转速150rpm和原位温度进行培养,并于培养开始后的第0天、1天、2天和4天分别陆续停止培养。
8.根据权利要求1所述,本方法优选添加20ml 2mol/L的KCl到相应培养时间的血清瓶中,于150rpm摇床震荡15min,将沉积物中的亚硝酸盐和硝酸盐提取到液相中,再经过离心过滤处理,收集上清液待测。
9.根据权利要求1所述,要求通过NO3 -(处理1)和NO2 -(处理2)的累积速率差来计算得出全程硝化速率。
10.根据权利要求1所述,要求本方法中,线性回归系数(R2)要求大于0.80。
11.根据权利要求1所述,根据AOB和AOA的代谢速率,计算全程硝化对氨氮的去除贡献率。
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CN116769671A (zh) * | 2023-07-19 | 2023-09-19 | 华北电力大学 | 一种富集氨氧化微生物的方法及富集氨氧化微生物制品 |
CN116769671B (zh) * | 2023-07-19 | 2024-05-10 | 华北电力大学 | 一种富集氨氧化微生物的方法及富集氨氧化微生物制品 |
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CN116769671B (zh) * | 2023-07-19 | 2024-05-10 | 华北电力大学 | 一种富集氨氧化微生物的方法及富集氨氧化微生物制品 |
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