Soil aggregates affect the legacy effect of copper pollution on the microbial communities

Soil aggregates provide spatially heterogeneous microhabitats for microorganisms and their activities. In metal contaminated soil, the microbial community is shaped by both metal stress and aggregate sizes. However, it remains unclear whether and how aggregate sizes affect the legacy effect of metal pollution on microbial communities. Here, we investigated the legacy effect of long-term copper (Cu) pollution on the distribution patterns of the bacterial and protistan communities in soil aggregates. Our results showed that soil aggregates of different sizes possessed distinct bacterial and protistan communities under Cu-polluted and unpolluted conditions (aggregate effects), as reflected by distinct α-diversity and community compositions. The bacterial and protistan communities in micro- and macro-aggregates were more responsive to Cu pollution than those in mega-aggregates, primarily because micro- and macro-aggregates contained a greater number of Cu susceptible bacterial and protistan species. Ciliophora and Chlorophyta displayed great sensitivity to Cu, especially in small aggregate-size fractions, and could serve as a bio-indicator of Cu pollution. Furthermore, the magnitude of aggregate effects on microbial communities was weakened in Cu-polluted soils compared to unpolluted soils. With decreasing aggregate sizes, the co-occurrence networks between bacteria and protists became more complex, connected and compact. Robustness analysis showed that phagotrophic protists played essential roles in sustaining the network stability, especially in mega-aggregates. Our findings highlight the asymmetrical changes in microorganisms in aggregates under long-term Cu pollution, providing a novel insight into the legacy effects of metal pollution on soil microbiomes at the micro-level.