BIOLOG微平板技术检测农田土壤微生物群落结构的方法比较

Comparison of different BIOLOG microplate technology for detecting carbon metabolism of farmland soils

以采自湖北省荆州市和黄冈市的农田土壤作为供试土样,分别采用土壤样品冰浴后上清液100倍稀释、冰浴后悬浊液1 000倍稀释和离心去碳源3种方法,利用多功能酶标仪间隔12h检测D值,并计算AWCD(average well color development,每孔平均颜色变化)值来探究分析土壤肥力高、富含有机质、可利用碳源丰富的农田土壤微生物代谢功能的BIOLOG-ECO微平板技术适宜方法。结果表明,土样冰浴后上清液100倍稀释的方法不能有效去除土壤中可利用碳源,不能准确反映土壤微生物对碳源的利用程度。而冰浴后悬浊液1 000倍稀释、离心去碳源都能准确显示土壤微生物的活性变化,但是两者AWCD的标准差有极显著的差异,比较而言,离心去碳源的方法能更稳定地检测土壤微生物群落的代谢能力。研究结果为利用BIOLOG-ECO微平板技术检测富含有机质的农田土壤微生物代谢功能多样性提供了有价值的信息。

Three methods of BIOLOG microplate analysis with BIOLOG ECO Plate were applied and compared to explore the optimal way to analyze the carbon metabolism of farmland soils with carbon abundance.The soil samples were collected from Jingzhou Municipality and Huanggang Municipality,Hubei,China.The 100-fold eluted supernatant was incubated after incubating on ice.The 1 000-fold eluted suspension was incubated after incubating on ice.The supernatant was centrifuged after removing carbon.Color development of each well with optical density at 595 nm and 750 nm every 12 hwas recorded with an ELISA Reader.The microbial activity in each microplate was expressed as average well color development(AWCD).We found that incubating 100-fold eluted supernatant could not accurately reflect the microorganism activities due to the available carbon in the supernatant which caused the reaction in the control well.The other two methods can clean up the carbon.The standard of AWCD of the method of incubating 1 000-fold eluted suspension after incubating on ice was significantly higher than that of incubating the supernatant after removing carbon with centrifuging,indicating that the latter one was more stably to detect the carbon metabolism of farmland soils.