Summary: To gain insight into microbial function following increased atmospheric CO ₂ concentration, we investigated the influence of 9 years of enriched CO ₂ (600 μl litre ⁻¹ ) on the function and structural diversity of soil microorganisms in a grassland ecosystem under free air carbon dioxide enrichment (FACE), as affected by plant species ( Trifolium repens L. and Lolium perenne L. in monocultures and mixed culture) and nitrogen (N) supply. We measured biomass and activities of enzymes covering cycles of the most important elements (C, N and P). The microbial community was profiled by molecular techniques of phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analysis. The enrichment in CO ₂ increased soil microbial biomass (+48.1%) as well as activities of invertase (+36.2%), xylanase (+22.9%), urease (+23.8%), protease (+40.2%) and alkaline phosphomonoesterase (+54.1%) in spring 2002. In autumn, the stimulation of microbial biomass was 25% less and that of enzymes 3–12% less than in spring. Strong correlations between activities of invertase, protease, urease and alkaline phosphomonoesterase and microbial biomass were found. The stimulation of microbial activity in the enriched atmosphere was probably caused by changes in the quantity and kind of root litter and rhizodeposition. The response of soil microorganisms to enriched CO ₂ was most pronounced under Trifolium monoculture and under greater N supply. The PLFA analysis revealed that total PLFA contents were greater by 24.7% on average, whereby the proportion of bioindicators representative of Gram‐negative bacteria increased significantly in the enriched CO ₂ under less N‐fertilized Lolium culture. Discriminant analysis showed marked differences between the PLFA profiles of the three plant communities. Shannon diversity indices calculated from DGGE patterns were greater (+12.5%) in the enriched CO ₂ , indicating increased soil bacterial diversity. We conclude that greater microbial biomass and enzyme activity buffer the potential increase in C sequestration occurring from greater C addition in enriched CO ₂ due to greater mineralization of soil organic matter.

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