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Root mucilage modulates soil-plant-water dynamics, but its interactions with microbial community functioning remain poorly understood. The aims of this study were to estimate (I) the impacts of mucilage and soil water content on the microbial community composition and (II) the mucilage consumption by individual microbial groups. C4 root mucilage from maize (at 40 and 200 μg C per gram dry soil, corresponding to 10 and 50% of soil microbial biomass, respectively) was added in single pulses to a C3 soil at two moisture levels: optimum (80% of water-holding capacity (WHC)) and drought (30% of WHC). After 15 days of incubation, the microbial community composition was studied by phospholipid fatty acids (PLFA) analysis and incorporation of mucilage-derived C into individual microbial groups was determined by compound-specific isotope analysis. Microbial community composition remained largely unaffected by mucilage addition but was affected by moisture. Whereas an increase in water content reduced mucilage C recovery in PLFA for the low-dose mucilage amendment from 19 to 9%, it had no effect under the high-dose amendment (11-12%). This suggests that the role of mucilage for microbial functioning is especially pronounced under drought conditions. The fungal PLFA 18:2ω6,9 was present only under drought conditions, and fungi profited in their mucilage C utilisation from the lower competitiveness of many bacterial groups under drought. In this study, Gram-negatives (G−, characterised by PLFA 18:1ω9c, 18:1ω7c, 16:1ω7c and cy17:0) showed the highest mucilage-derived C in PLFA, especially at the high-dose amendment, suggesting them to be the major decomposers of mucilage, especially when the availability of this C source is high. Gram-positives (G+) included different sub-groups with distinct responses to moisture: G+ 1 (a15:0) were only competitive for mucilage C under drought, whereas G+ 3 (i17:0) were only able to utilise mucilage-derived C under optimal moisture conditions. During the 15-day incubation, they built up more than 40% of their membranes from mucilage-derived C, suggesting that in the case of high availability, mucilage can act as an important C source for this microbial group. However, under drought, G− 1 and fungi were incorporating the most mucilage C into their membranes (approx. 20% of PLFA-C). The observation that, for some groups, the high-dose mucilage amendments under drought led to higher C incorporation into PLFA than under optimum moisture suggests that mucilage can compensate drought effects for particular microbial groups. Thus, mucilage may not only act as a C source for microorganisms but may also mitigate drought effects for specific rhizosphere microbial groups. [ABSTRACT FROM AUTHOR]