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• Microbial communities in groundwater before and after the earthquake are compared. • Relation between water drawdown and microbial community change is discussed. • Recovery process of microbial community after the earthquake is studied. • Changes of denitrifying bacterial community is also observed. Post-seismic microbial changes in aquifer systems have rarely been documented, and the processes to explain these changes have yet to be explicated. Microbial communities within groundwater in the Kumamoto area, Japan were characterized in 2013 and 2014 prior to the occurrence of a large earthquake (7.0 Richter scale) in 2016, which provided a rare opportunity to compare samples obtained following the seismic activity, with information from the previous, and subsequent years 2017 and 2018. Microbial changes were monitored for 4 representative wells in a regional groundwater flow system utilizing a previously proposed post-seismic hydrogeological change model. Site-specific microbial communities changed and became homogeneous for all of the wells after the earthquake. The original microorganisms such as methylotrophs had decreased; however, exogenous microorganisms, such as Propionibacterium acnes , increased at all sites in the 2–3 months following the earthquake, these changes were consistent with the coseismic surface water drawdown model. However, the prevalence of P. acnes clones decreased in the 2–3 years following the earthquake. Notably, none of the microbial communities recovered to their original condition prior to the earthquake. This suggested that a new water supply from mountain aquifers could have caused these second stage post-seismic microbial changes. Our study demonstrated that large crustal earthquakes could dramatically alter microbial communities within aquifer systems caused by post-seismic hydrogeological changes. However, future monitoring is required to reveal further changes during the microbiological recovery process. [ABSTRACT FROM AUTHOR]