Methodological estimation of the contribution of microbial living biomass carbon to soil organic carbon in grasslands

Accurate quantification of the soil microbial living biomass carbon pool is critically important for advancing research on the key processes of microbial-mediated soil carbon transformation. Previous research has widely recognized the entombing effect of microbial necromass carbon is essential to soil organic carbon (SOC) sequestration, whereas the contribution of microbial living biomass carbon to SOC is often overlooked because of the small carbon reservoir capacity, rapid turnover rate, and the complexity of isolation and quantification. Meanwhile, the establishment of a universal microbial living biomass carbon quantification framework is urgently needed due to challenges of the absence of widely applicable methodologies and carbon conversion coefficients. This limitation impedes our understanding of the pivotal role of microbial living biomass in the formation mechanisms, turnover processes, and stability maintenance of SOC. Here, the accuracy and applicability of microbial living biomass carbon were compared by flow cytometry (FCM), propidium monoazide combined with qPCR (PMA-qPCR) and phospholipid fatty acids (PLFA) methods combined carbon conversion coefficient in four distinct soil types from the Inner Mongolian grasslands. The results showed that: (1) the estimation of bacterial (385 μg/g vs. 568 μg/g, R=0.46), fungal (88 μg/g vs. 103 μg/g, R=0.44) and total (473 μg/g vs. 671 μg/g, R=0.48) microbial living biomass carbon by PLFA and PMA-qPCR methods showed good repeatability and significant correlations, indicating the reliability of the conversion coefficients used in these methods. A significant positive association was observed between the estimation of bacterial living biomass carbon by FCM with both PMA-qPCR and PLFA, still, it was 1‒3 orders of magnitude lower than the other two methods, suggesting the need for improved practical application of FCM with its associated conversion coefficient. The relative deviation from different methodologies was positively impacted by SOC, soil total nitrogen, cation exchange capacity, and soil clay and silt content. (2) Overall, microbial necromass carbon is 7.28‒15.81 times of living biomass carbon, and plant biomass, soil physicochemical properties and microbial life strategies significantly influence the accumulation dynamics and ratio of necromass carbon and living biomass carbon. Bacterial living biomass carbon is 3.43‒4.99 times higher than fungi, while bacterial necromass carbon is only 0.32‒0.38 times that of fungi. These ratios indicate that higher living biomass carbon content did not necessarily equate to greater accumulation of necromass carbon, underscoring the necessity for future research on the turnover of living biomass and necromass carbon. (3) Compared to microbial necromass carbon, living biomass carbon was more significantly associated with particulate organic carbon formation and CO₂ release, and necromass carbon was strongly associated with SOC and mineral-associated organic carbon accumulation. These findings are scientifically significant for establishing a quantitative estimation system for microbial living biomass carbon, understanding the critical role of biomass turnover in soil carbon sequestration and stability, and optimizing parameters in the carbon cycle model.