Zhang Shanshan, Jiao Xinyu, Kang Hongzhang, Yu Wenjuan
State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou, China.
College of Chemistry and Materials Engineering, Zhejiang Agriculture and Forestry University, Hangzhou, China.
Front Microbiol. 2025 May 14;16:1587425. doi: 10.3389/fmicb.2025.1587425. eCollection 2025.
Phospholipid fatty acid (PLFA) method has been popular and powerful for characterizing soil microbial communities over decades. However, little is known about efficiencies of acidic vs. alkaline extractants and catalysts in PLFA extraction and methylation. More urgently, it remains unclear whether methanol could efficiently elute phospholipids while avoiding recovering non-target lipids. Here, by adding pure lipid standards representative of major neutral, glyco-, and phospholipids into acidic and alkaline soils collected from subtropical China, we comprehensively evaluated efficiencies of extraction, elution, and methylation of three steps in the PLFA method. A good proportion of PLFAs could be extracted by phosphate buffer with both acidic (42-51%) and alkaline (43-68%) soils while citrate buffer worked better for acidic (43-46%) than alkaline (36-47%) soils. Phospholipids were expected to be mainly eluted in methanol, yet we found a non-negligible proportion of phospholipids eluted by chloroform for both acidic (36-71%) and alkaline (9-55%) soils, which is much larger than previously reported. Only 42-50% (acidic soils) and 45-68% (alkaline soils) of phospholipids were recovered in methanol. Meanwhile, 16% (acidic soils) and 5% (alkaline soils) of glycolipid DGDG were unexpectedly eluted into methanol. The alkaline catalyst (mean 86% across all investigated phospholipids) was more efficient in facilitating phospholipids methylation than the acidic one (mean 67%). Overall, incomplete separation among lipid types caused loss of phospholipids and introduction of glycolipid interferences in the methanol fraction, leading to biased estimation of soil microbial biomass and composition. Chloroform elution of phospholipids challenges the principle of "like dissolves like" in solid-phase chromatography, although we cannot rule out the uncertainty caused by background PLFAs and future experiments are needed to provide more evidence. To effectively remove lipid interferences and efficiently elute phospholipids, possible solutions include replacing chloroform with hexane, increasing elution volumes of acetone and methanol, and/or using anion exchange columns.
几十年来，磷脂脂肪酸（PLFA）方法在表征土壤微生物群落方面一直很流行且功能强大。然而，关于酸性和碱性萃取剂及催化剂在PLFA提取和甲基化中的效率，人们了解甚少。更迫切的是，甲醇能否在避免回收非目标脂质的同时有效地洗脱磷脂仍不清楚。在此，我们通过将代表主要中性脂质、糖脂和磷脂的纯脂质标准品添加到从中国亚热带地区采集的酸性和碱性土壤中，全面评估了PLFA方法中提取、洗脱和甲基化三个步骤的效率。相当一部分PLFAs可以用磷酸盐缓冲液从酸性土壤（42 - 51%）和碱性土壤（43 - 68%）中提取出来，而柠檬酸盐缓冲液对酸性土壤（43 - 46%）的效果比对碱性土壤（36 - 47%）更好。预计磷脂主要在甲醇中洗脱，但我们发现，对于酸性土壤（36 - 71%）和碱性土壤（9 - 55%），氯仿洗脱的磷脂比例不可忽略，这比之前报道的要大得多。在甲醇中回收的磷脂仅占42 - 50%（酸性土壤）和45 - 68%（碱性土壤）。与此同时，糖脂二半乳糖甘油二酯（DGDG）有16%（酸性土壤）和5%（碱性土壤）意外地洗脱到甲醇中。碱性催化剂（所有研究的磷脂平均为86%）在促进磷脂甲基化方面比酸性催化剂（平均67%）更有效。总体而言，脂质类型之间的不完全分离导致了磷脂的损失以及甲醇部分中糖脂干扰物的引入，从而导致对土壤微生物生物量和组成的估计出现偏差。磷脂在氯仿中的洗脱对固相色谱中“相似相溶”的原则提出了挑战，尽管我们不能排除背景PLFAs造成的不确定性，还需要未来的实验提供更多证据。为了有效去除脂质干扰并有效地洗脱磷脂，可能的解决方案包括用己烷代替氯仿、增加丙酮和甲醇的洗脱体积，和/或使用阴离子交换柱。
