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Environment
模拟土壤孵育实验中的温度

Research Article

模拟土壤孵育实验中的温度

DOI: 10.3791/64081

October 28, 2022

Jianwei Li1, Precious Areeveso1, Xuehan Wang1, Siyang Jian1,2, Lahiru Gamage1

1Department of Agricultural and Environmental Sciences,Tennessee State University, 2Department of Plant Biology and Microbiology,University of Oklahoma, Norman

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In This Article

Summary Abstract Introduction Protocol Representative Results Discussion Disclosures Acknowledgements Materials References Reprints and Permissions

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Retraction Notice

The article Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data (10.3791/61715) has been retracted by the journal upon the authors' request due to a conflict regarding the data and methodology. View Retraction Notice

Summary

实验室土壤变暖实验通常在多个腔室中使用两个或多个恒定温度。通过提出一个复杂的环境箱,我们提供了一种精确的温度控制方法来模拟 原位 土壤温度的大小和幅度,并改进土壤培养研究的实验设计。

Abstract

研究变暖对土壤的影响需要真实准确地表示温度。在实验室培养研究中,一种广泛采用的方法是在多个腔室中呈现恒定温度,并通过比较低温和高温室之间的土壤响应, 得出 变暖对土壤变化的影响。然而,这种常用的方法未能模仿在现场条件下观察到的实际温度的大小和振幅,因此可能破坏此类研究的有效性。随着复杂的环境箱越来越多,必须研究土壤培养研究的替代温度控制方法。该协议将引入最先进的环境室,并演示传统的和新的温度控制方法,以改进土壤培养的实验设计。该方案主要包括四个步骤:温度监测和编程、土壤收集、实验室孵化和变暖效应比较。将举一个例子来演示不同的温度控制方法以及由此产生的对比变暖情景;即称为逐步升温(SW)的恒温设计,以及模拟的 原 位温度设计为逐渐变暖(GW),以及它们对土壤呼吸,微生物生物量和胞外酶活性的影响。此外,我们还提出了一种使温度变化情景多样化的策略,以满足特定的气候变化研究需求(例如,极端高温)。温度控制协议和推荐的量身定制和多样化的温度变化场景将有助于研究人员在实验室中建立可靠和现实的土壤培养实验。

Introduction

预计本世纪全球地表温度将上升1.8-6.4°C1,2。全球变暖可能会增加从土壤到大气的CO2通量,导致变暖3,4,5,6的正反馈。由于微生物群落在调节土壤对变暖的呼吸响应中起着关键作用7,8,微生物呼吸的变化及其随变暖的潜在微生物机制一直是研究重点。尽管在现场条件下通过加热电缆9和开顶室10部署的土壤增温实验有利于捕获温度11等自然土壤特征,但其高昂的安装和维护成本限制了其应用。或者,不同温度的土壤培养实验是一个不错的选择。在实验室中土壤培养的主要优点是,控制良好的环境条件(例如温度)能够在现场实验环境中将单因素效应与其他混杂因素分开12,13。尽管生长室和田间实验(例如植物生长)之间存在差异,但从实验室结果到田间的结果转换是很容易获得的 14.在实验室环境中孵育土壤样品可以帮助提高我们对土壤对变暖响应的机制理解15。

我们的文献综述确定了几种温度控制方法,因此在过去的土壤孵化研究中确定了不同的温度变化模式(表1)。首先,用于控制温度的仪器大多通过培养箱、生长室、水浴,在极少数情况下,还通过加热电缆。根据这些仪器,已经产生了三种典型的温度变化模式(图1)。其中包括实现最多的模式,恒温(CT),具有非零恒温变化率的线性变化(LC)和具有昼夜温度类型的非线性变化(NC)。对于CT模式的情况,温度可能会随着时间的推移而变化,尽管恒温在孵育期间保持一定时间(图1B)。对于LC,不同研究中的温度变化率可能变化超过两个数量级(例如,0.1°C/天与3.3°C/h; 表1);对于NC案例,大多数依赖于所用仪器的内在容量,从而导致各种模式。尽管通过加热电缆或培养箱声称了一种昼夜温度变化16,17;然而,这些实验中的腔室温度未经验证。 表1 中的其他主要审查结果包括孵育温度范围为0-40°C,大多数在5-25°C之间;实验持续时间从几小时(<1天)到近2年(~725天)不等。此外,从森林、草原和农田生态系统中收集了受孵化的土壤,其中主要分布在美国、中国和欧洲,主要分布在美国、中国和欧洲(表1)。

鉴于三种主要的温度变化模式, 表2总结了过去研究中实现的几种不同的变暖情景。它们包括逐步变暖(SW),不同幅度的SW(SWv),线性逐渐变暖(GWl),非线性逐渐变暖(GWn)和昼夜逐渐变暖(GWd)。

总之,过去的土壤孵化通常捕获一个地点的平均空气或土壤温度。在许多情况下,如表1所示,培养箱或培养箱在固定温度下手动编程,但不能根据需要自动调节温度,缺乏控制温度随时间变化的模式和速率的能力(方程1),因此难以模仿当地土壤的昼夜温度。另一方面,尽管在两个实验中进行了尝试16,17,但我们没有发现在其孵化实验中明确模仿昼夜逐渐变暖(GWd)的研究(表1)。根据文献综述,主要障碍在于糟糕的实验设计,特别是缺乏能够实施和验证昼夜或其他逐渐变暖情景的复杂仪器。

Equation 1(公式1)

其中ΔT 是温度变化的量, m 是温度变化的模式, r 是温度变化的速率, t 是变化的持续时间。

为了提高土壤培养实验的严谨性,该文提出一种准确、精密的温度控制方法。新设计采用最先进的环境室,越来越可用且经济可行,不仅能够准确模拟 原位 土壤温度(例如昼夜模式),而且通过考虑可能的极端温度变化,提供一种可靠的方法来最大限度地减少仪器偏差的伪像。目前的土壤培养设计应帮助研究人员确定满足其培养和研究需求的最佳策略。该方法的总体目标是为土壤生物地球化学家提供一种高度可操作的方法来改革土壤孵化设计。

Protocol

1. 温度监控和编程

  1. 确定研究地块内的采样区域。在10厘米深度的土壤中安装一个或几个自动温度探头。通过数据传输电缆 将 气象站连接到计算机,然后打开计算机上的软件。
  2. 单击 "启动/属性 "工具栏按钮,为正在使用的外部传感器配置记录器。
  3. 在 "属性 "屏幕上,设置记录器/工作站名称(即土壤培养经验)和数据收集间隔(即 60 分钟)。然后,在" 属性 "屏幕上,单击正在使用的外部传感器端口上的 "启用" ,然后从每个传感器端口(即端口 A;"启用":温度°C)。最后,单击 "确定 "保存设置。
  4. 每周监控探头的读数以避免故障,并每月下载一次数据集。获得涵盖生长季节(即 4 月至 9 月)的几个月的完整记录。
  5. 对温度记录进行数据分析。通过对所有观测值求平均值来获取生长季节的平均每小时温度。
    1. 通过在生长季节所有日子中平均同一小时的温度来获得每天每小时的平均温度。
  6. 在复杂的腔室中,启动软件并单击主菜单屏幕上的 "配置文件 "按钮以创建新文件。在文件名输入行中,输入"SW low"。通过单击 即时更改 选项,输入 15.9 °C 作为步骤 1.5 中获得的初始温度,然后在分钟行中输入 2 以保持温度 2 分钟 ,然后单击 完成 按钮。然后,在 斜坡时间 选项下,输入 15.9 °C 作为目标设定点,并在 小时 行输入 850 小时以维持温度。最后,点击 完成 按钮。
    1. 在第二个腔室中重复上述步骤,向每个温度节点添加5°C,并创建一个新的文件名"SW high"。
    2. 在第三个腔室中重复步骤1.4,添加23个额外的步骤,对应于步骤1.5.1中获得的23个观察到的每小时土壤温度。在最后一步,称为 JUMP,设置42个重复循环(跳转计数42)。这导致了逐渐变暖或GW低的情况。
    3. 在第四个腔室中重复上述步骤,向每个温度节点添加5°C。这将允许在更高的温度水平(即GW高)下模拟42天的变化温度。
  7. 进行24小时的初步运行,并输出四个腔室记录的温度。将腔室记录的温度与编程的温度绘制成图表(图2A-D)。
    1. 如果在24小时内达到的温度与通过温差<0.1°C编程的温度相匹配(图2A,B,E,F),则腔室适用于土壤培养实验。
    2. 如果在这些腔室中的任何一个中不满足标准,请重复另一个24小时测试或寻找新的腔室。

2. 土壤收集和均质化

  1. 在温度探头区域附近,收集5个0-20厘米深度的土壤样本,去除表面凋落物层后放入一个塑料袋中。
  2. 通过扭曲、压制和混合袋中的材料来彻底混合样品,直到看不到单个土壤样品。
  3. 将样品储存在装满冰袋的冷却器中,并立即将样品运送到实验室。
  4. 去除每个岩心中的根部,将其通过2毫米的土壤筛筛过筛,并在进行后续分析之前彻底混合和均质化样品。

3. 实验室培养

  1. 孵育前,称取10.0g新鲜土壤,在105°C下烘干24小时,称量干燥土壤。推导出新鲜土壤和干燥土壤样本之间的差异,并计算与干燥土壤重量的差异比率,以确定电子表格中的土壤水分含量。
  2. 使用派生的含水量计算土壤微生物生物量碳 (MBC)、胞外酶活性 (EEA) 和土壤异养呼吸,如以下步骤所述。这些数据将有助于了解处理对土壤呼吸的影响以及潜在的微生物机制。
  3. 在孵育之前,称量田间潮湿的土壤子样品(10g),并通过氯仿熏蒸-K2SO4 提取和过硫酸钾消化方法定量土壤MBC。
  4. 在孵育之前,称量田间潮湿土壤的子样品(1.0 g)并测量土壤水解和氧化EEA19。
  5. 在底部用玻璃纤维纸密封的 16 个聚氯乙烯 (PVC) 芯(直径 5 厘米,高 7.5 厘米)中称量 16 个田间潮湿土壤子样品(相当于干重 15.0 克)。
  6. 将PVC芯放入内衬玻璃珠床的梅森罐(~1 L)中,以确保芯不会吸收水分。
  7. 如步骤1.4所述,在四个腔室中分别放置四个罐子。打开腔室并在四个腔室中同时启动程序。
  8. 在孵育期间,在第2小时,第1,2,7,14,21,28,35和42天,将所有罐子放在四个腔室中的每一个中,并使用便携式CO2 气体分析仪通过将分析仪的项圈放在每个罐子的顶部来测量土壤呼吸速率(Rs)。
  9. 在孵育结束时(即第42天)破坏性地收集所有罐子并量化土壤MBC,如步骤3.3中所述。
  10. 在孵育结束时(即第42天)破坏性地收集所有罐子,并如步骤3.4中所述量化土壤酶活性。

4. 增温效果比较

  1. 通过假设两个连续集合之间的呼吸速率 (Rs) 恒定,使用呼吸速率乘以持续时间来得出累积呼吸 (Rc)。
  2. 进行三向重复测量方差分析(ANOVA),以测试时间,温度(变暖)和温度模式(变暖情景)对Rs 和Rc的主要和交互效应。此外,进行双向方差分析以测试变暖和变暖情景对MBC和EEA的影响。

Representative Results

所选最先进的腔室以高精度复制目标温度(图2A,B,E,F),并满足孵育实验的技术要求。鉴于易于使用和操作,这标志着在土壤变暖研究和其他应用(如植物研究)中改进温度模拟的技术。该程序已被用于我们最近基于田纳西州中部柳枝稷农田的案例研究中。

研究结果表明,相对于对照治疗,在两种变暖情景(SW和GW)中,变暖导致呼吸损失(Rs和R c)显着增加,并且GW使变暖引起的呼吸损失(Rc)相对于SW翻了一番,分别为81%和40%(图3)。在第42天,西南和GW之间的MBC和EEA也存在显着差异,因此SW中的MBC高于GW(69%对38%;图4)GW中的糖苷酶和过氧化物酶(例如,AG,BG,BX,CBH,NAG,AP,LAP)明显高于SW情景(图5)。

Figure 1
图1:土壤变暖实验中温度变化模式的图示,概念见表1。 (A)大多数研究采用的恒温(CT)。(B)具有不同幅度的恒定温度(CTv)。(中,四)具有正负速率的线性变化 (LC)。(中、女)具有不规则模式和昼夜模式的非线性变化(NC)。请点击此处查看此图的大图。

Figure 2
图 2:在 24 小时测试期间通过编程 和腔室温度确定的温度。 (A,B)受控下的目标温度(灰线)和腔室温度记录(虚线)和逐步升温(SW)的升温处理;(中,四)控制下的目标温度(灰线)和腔室温度记录(虚线)和逐渐升温(GW)的升温处理;(东、 女)图 C 和 D中记录的温差。 请点击此处查看此图的大图。

Figure 3
图 3:在 42 天的土壤培养实验中,SW 和 GW 在控制(空心)和变暖(暗)处理下的平均 (± 标准误差) 累积土壤呼吸速率(Rc, μg CO2-C·g土壤-1)。插图显示了用于估计累积呼吸的土壤呼吸速率(R s,μg CO2-C·h-1·g soil-1),假设R s在下一次测量之前是恒定的。(A)逐步变暖(SW)和(B)逐渐变暖(GW)。N = 4 在每个集合中。请点击此处查看此图的大图。

Figure 4
图 4:在 42 天的土壤培养实验中,SW 和 GW 在控制和加温处理下的平均 (± 标准误差) MBC。 MBC = 微生物生物质碳;N = 4 在每个集合中。S表示基于三向重复测量方差分析的变暖情景(SW与GW)的显着影响, p < 0.05。 请点击此处查看此图的大图。

Figure 5
图 5:在 42 天的孵育实验中,对照和加温处理下 SW 和 GW 中的平均 (± SE) 糖苷酶和过氧化物酶(μmol活性 h-1·gsoil-1)。 BX =β1,4-木糖苷酶;AP =酸性磷酸酶;圈数 =亮氨酸氨基肽酶;NAG =β-1,4-N-乙酰氨基葡萄糖苷酶;牛 =氧化酶; 磷酸 =苯酚氧化酶;每 =过氧化物酶。N = 4 在每个集合中。S表示变暖情景(SW与GW)的显着影响,基于三向重复测量方差分析,p < 0.05。请点击此处查看此图的大图。

表1:土壤培养研究中温度控制方法和温度变化模式的文献综述 12,13,16,17,20,21,22,23,24,25,26,27,28,29, 30,31,32,
33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51、
52,53,54,55,56,57,58,59,60,61,62。

该评价共纳入46项研究。 请按此下载此表格。

表2:基于文献综述的主要温度变化模式和相应的变暖情景(表1)。 建立了五种模式和情景来表示各种可能的温度变化和变暖条件。请按此下载此表格。

Discussion

作者没有什么可透露的。

Disclosures

实验室土壤变暖实验通常在多个腔室中使用两个或多个恒定温度。通过提出一个复杂的环境箱,我们提供了一种精确的温度控制方法来模拟 原位 土壤温度的大小和幅度,并改进土壤培养研究的实验设计。

Acknowledgements

用于支持该研究的资金来源包括美国国家科学基金会 (NSF) HBCU−EiR(第 1900885 号)、美国农业部 (USDA) 农业研究服务 (ARS) 1890 年代教师研究休假计划(编号 58-3098-9-005)、美国农业部 NIFA 拨款(编号 2021-67020-34933)和美国农业部埃文斯-艾伦拨款(第 1017802 号)。我们感谢田纳西州纳什维尔TSU主校区农业研究和推广中心(AREC)工作人员的帮助。

Materials

,,的 FilterMax F5用于土壤收集 用于的
10 mL 注射器Fisher Scientific14-826-13用于土壤呼吸测量
Composer 软件TestEquity型号 #107,用于培养温度设置
环境室TestEquity型号 #107,用于土壤培养
环境气体分析仪PP SystemsEGM5用于土壤呼吸测量
滤纸Fisher Scientific1005-125用于土壤培养
梅森罐球15381-3用于土壤培养
烘箱Fisher Scientific15-103-0520用于土壤水分测量
塑料拉链密封储物袋Fisher Scientific09-800-16用于土壤收集
读板器 分子设备用于土壤细胞外酶分析
R 软件R FoundationR 版本 4.1.3 (2022-03-10)用于统计计算
冰箱/冰柜Fisher Scientific13-991-898用于土壤储存
螺丝刀Fisher Scientific19-313-447用于土壤收集
SharpieFisher Scientific50-111-3135用于土壤收集
SieveFisher Scientific04-881G 用于筛分土壤样品
硅胶隔垫Duran Wheaton kimble224100-070用于土壤培养的梅森罐
的土壤螺旋钻AMS350.05用于土壤收集
SpecWare 软件Spectrum TechnologiesWatchDog E2700 (3340WD2) 用于温度收集间隔设置
温度探头Spectrum TechnologiesWatchDog E2700 (3340WD2) 用于土壤温度测量
TOC/TN 分析仪岛津土壤微生物生物量分析TOC-L 系列

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模拟土壤孵育实验中的温度
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