Extracellular Enzyme Activity data

Read me: Measurement of Extracellular Enzyme Activity data in the soil incubation study in summer 2018. A detailed description of methods used to derive the data is below. Further questions can be directed to [e-mail] Publication : Adaptive Multi-Paddock Grazing Lowers Soil Greenhouse Gas Emission Potential by Altering Extracellular Enzyme Activity Authors: Shrestha, Bharat M.; Bork, Edward W.; Chang, Scott X.; Carlyle; Cameron N.; Ma, Zilong, Döbert, Timm F.; Kaliaskar, Dauren; Boyce, Mark S. Published in: Agronomy DOI: doi:10.3390/agronomy10111781 Methods: Measurements of microbial activities and soil parameters A parallel set of soils at the same moisture level were prepared by placing 50 g of oven-dry equivalent air-dried soil in 200 mL conical flasks for measuring extracellular enzyme activities (EEAs), microbial biomass C (MBC) and N (MBN), and reactive N (available-N), on day 1 (start), day 13, and day 102 (end) of the incubation period. Activities of select extracellular enzymes involved in C (xylosidase: Xylo, β-glucosidase: BG, cellobiosidase: Cello) and N (N-acetyl-β glucosaminidase: NAC) cycling in soil were analyzed. To assess the EEA, a standard fluorometric method was used with 96-well microplates (see Sinsabaugh et al. [46]) with acetate buffer solution (pH 5.0). One gram of fresh soil and 125 mL of buffer were mixed to make a soil solution and 200 µL of the solution was pipetted into each well of the microplate. Depending on the enzyme type, microplates with soil solutions and enzyme substrates were incubated for three (BG, NAC), four (Xylo), or seven hours (Cello) at 25 °C. After incubation, microplates were read on a Biotek Synergy HT (BioTek Instruments, Inc., Vermont, USA) with 360 nm excitation and 460 nm emission [47]. Substrates used in this experiment were 4-MUF-β-D-glucopyranoside, 4-MUF-β-D-cellobioside, 4-MUF-β-D-xyloside, and 4-MUF-N-acetyl-β-glucosaminide. Soil MBC and MBN were analyzed by the chloroform fumigation-extraction method [48,49]. For fumigation, 10 g of moist soil sample was fumigated with chloroform in a desiccator for 24 h. Soil extracts were obtained by mixing 10 g of moist soil with 50 mL of 0.5 mol L−1 K2SO4 solution, shaking for 1 h in a reciprocating shaker (250 rpm) and filtering through Q2 filter papers. Soil extractions were analyzed for MBC and MBN by a TOC-V analyzer connected to a TN module (Shimadzu Corporation, Kyoto, Japan). The MBC and MBN were calculated as the difference between the C and N extracted from fumigated and non-fumigated soil samples, respectively. Soil NO3- and NH4+ were determined using the colorimetric method in soil solution. The vanadium oxidation method was used for NO3- [50], and the indophenol blue method was used for NH4+ [51] and analyzed on a spectrophotometer (GENESYS™ 10S UV-Vis Spectrophotometer, ThermoFisher Scientific, USA ). The sum of NH4+-N and NO3--N was expressed as total available N (avail-N). The MBC, MBN and avail-N on each sampling day were calculated per unit mass of soil (mg kg-1 soil).

说明文档：2018年夏季土壤培养试验中胞外酶活性（extracellular enzyme activity, EEA）测定数据集。下文详细阐述了该数据集的获取方法。如有进一步疑问，可联系邮箱[e-mail]。
发表文献：《适应性多围栏放牧通过改变土壤胞外酶活性降低土壤温室气体排放潜能》
作者：Shrestha, Bharat M.; Bork, Edward W.; Chang, Scott X.; Carlyle, Cameron N.; Ma, Zilong, Döbert, Timm F.; Kaliaskar, Dauren; Boyce, Mark S.
发表期刊：《Agronomy》，DOI：doi:10.3390/agronomy10111781

试验方法：微生物活性与土壤参数测定
本试验设置平行土壤样品，维持相同含水率：称取相当于50 g烘干土重的风干土壤，置于200 mL锥形瓶中，分别于培养周期第1天（起始）、第13天及第102天（结束）测定胞外酶活性（EEA）、微生物生物量碳（microbial biomass C, MBC）与微生物生物量氮（microbial biomass N, MBN），以及有效态氮（available-N）。
本试验分析了参与土壤碳循环（木糖苷酶：xylosidase, Xylo；β-葡萄糖苷酶：β-glucosidase, BG；纤维二糖水解酶：cellobiosidase, Cello）与氮循环（N-乙酰-β-氨基葡萄糖苷酶：N-acetyl-β glucosaminidase, NAC）的选定胞外酶活性。
胞外酶活性测定采用标准荧光光度法，以96孔微孔板为载体（参照Sinsabaugh等[46]的方法），使用乙酸缓冲液（pH 5.0）。称取1 g新鲜土壤与125 mL缓冲液混合制成土壤悬浮液，随后用移液枪向每个微孔中加入200 μL该悬浮液。根据酶种类不同，含土壤悬浮液与酶底物的微孔板需在25 ℃下分别培养3 h（BG、NAC）、4 h（Xylo）或7 h（Cello）。培养结束后，使用Biotek Synergy HT多功能酶标仪（美国佛蒙特州BioTek仪器公司）在激发波长360 nm、发射波长460 nm条件下读取荧光信号[47]。本试验所用底物为4-甲基伞形酮基-β-D-吡喃葡萄糖苷、4-甲基伞形酮基-β-D-纤维二糖苷、4-甲基伞形酮基-β-D-木糖苷与4-甲基伞形酮基-N-乙酰-β-D-氨基葡萄糖苷。
微生物生物量碳（MBC）与微生物生物量氮（MBN）采用氯仿熏蒸浸提法测定[48,49]。熏蒸步骤为：称取10 g湿润土壤样品置于干燥器中，用氯仿熏蒸24 h。浸提步骤为：称取10 g湿润土壤与50 mL 0.5 mol·L⁻¹ K₂SO₄溶液混合，在往复式摇床（250 rpm）振荡1 h，随后通过Q2滤纸过滤得到土壤浸提液。采用搭载总氮模块的TOC-V分析仪（日本岛津公司，京都）测定浸提液中的碳、氮含量。MBC与MBN分别通过熏蒸与未熏蒸土壤样品浸提得到的碳、氮含量差值计算得到。
土壤硝态氮（NO₃⁻-N）与铵态氮（NH₄⁺-N）采用比色法测定：硝态氮采用钒氧化还原法[50]，铵态氮采用靛酚蓝比色法[51]，使用GENESYS™ 10S紫外-可见分光光度计（美国赛默飞世尔科技公司）完成分析。将NH₄⁺-N与NO₃⁻-N含量之和计为总有效态氮（avail-N）。
各采样日的MBC、MBN与avail-N均以单位干重土壤计（mg·kg⁻¹ 干土）。