Geological activity shapes the microbiome in deep-subsurface aquifers by advection

Abstract

Subsurface environments host diverse microorganisms in fluid-filled fractures; however, little is known about how geological and hydrological processes shape the subterranean biosphere. Here, we sampled three flowing boreholes weekly for 10 mo in a 1478-m-deep fractured rock aquifer to study the role of fracture activity (defined as seismically or aseismically induced fracture aperture change) and advection on fluid-associated microbial community composition. We found that despite a largely stable deep-subsurface fluid microbiome, drastic community-level shifts occurred after events signifying physical changes in the permeable fracture network. The community-level shifts include the emergence of microbial families from undetected to over 50% relative abundance, as well as the replacement of the community in one borehole by the earlier community from a different borehole. Null-model analysis indicates that the observed spatial and temporal community turnover was primarily driven by stochastic processes (as opposed to deterministic processes). We, therefore, conclude that the observed community-level shifts resulted from the physical transport of distinct microbial communities from other fracture(s) that outpaced environmental selection. Given that geological activity is a major cause of fracture activity and that geological activity is ubiquitous across space and time on Earth, our findings suggest that advection induced by geological activity is a general mechanism shaping the microbial biogeography and diversity in deep-subsurface habitats across the globe.

Keywords: deep subsurface; fractured aquifers; microbial biogeography; microbial community; microbial transport.

Fig. 1.

Location, borehole configuration, and permeable fracture network at the deep-subsurface field site. (A) Field site located at the SURF along the west drift 1478 m below ground surface. (B) Example of a core log photo illustrating the difference among rock matrix, a sealed fracture, and an open (natural) fracture: The permeability of an open fracture is orders of magnitude larger than a sealed fracture/rock matrix. (C) Borehole configuration of the field site showing all boreholes at view-1 (Top) and view-2 (Bottom). (D) Simplified conceptual model of the fracture network in the crystalline-rock formation, modified from Wu et al. (72). Gray and red circles represent the packer intervals in boreholes I (“Inj”) and P (“PI”), whereas the brown circle represents the segment in borehole P below the packer interval (“PB”). Realistic representations of the natural and hydraulic fractures can be found in Zhang et al. (8) and Schoenball et al. (73), respectively.

Fig. 2.

Changes in microbial community composition in the outflow from boreholes PDT, PST, and P over the 282-d sampling, with the injectate community composition included as a reference. (A) Industrial water injection into borehole I at a constant volumetric rate of 400 mL/min (except in the case of field operational problems, which paused the injection briefly). (B) Volumetric flow rate produced from each of the four sampling locations—PDT, PST, PI (borehole P within packer interval), and PB (borehole P below packer interval)—along with the total production rate record. “A”, “B” and “C” refer to the spontaneous flow rate change events on day 13, day 62, and day 154, as described in the Results. (C–F) Temporal dynamics of microbial community composition in produced fluids from PDT (C), PST (D), PI (E), and PB (F). (G) The microbial community composition in the injectate taken every day that a set of produced-fluid samples were obtained. Bar plots show the finest classification possible down to the family level. The major taxa (i.e., taxa that were within the top 10 most abundant in at least one sample) are shown in color. Legend is simplified to annotate only a subset of taxa in the produced fluids. See full legend in SI Appendix, Fig. S3.

Fig. 3.

PCoA on the microbial community data in the producing boreholes from day 0 to day 148, based on weighted Unifrac distance. (A) PCoA plot of the microbial community in produced fluids, with the PDT trajectory highlighted with black arrows. (B–D) The same PCoA plot as in (A) but highlighting the trajectory of PST (B), PI (C), and PB (D) using black arrows. Day 0 and the sampling dates revealing abrupt changes in microbial community composition are indicated next to the corresponding marker, highlighted with a black outline. Only data of the first 148 d and from the producing boreholes are included in this PCoA for ease of visualization. PCoA of the entire 282-d sample set along with the injectate is shown in SI Appendix, Fig. S4. Visual proximities of points are consistent with the optimal number of clusters defined using R function NbClust() (see Materials and Methods for details). PERMANOVA showed significant differences among the three defined clusters (pseudo-F = 46.66, R² = 0.42, P < 0.001), as shown in SI Appendix, Fig. S5.

Fig. 4.

Heatmaps representing RC_bray values for single-port (PDT) and cross-port (PI-PB) pairwise sample comparisons. (A) RC_bray values for pairwise comparisons among PDT samples, revealing the switch in assembly mechanism from homogenizing dispersal (pink) to dispersal limitation (green) over time (e.g., box 1 to box 2), consistent with advective mixing during the sampling period due to fracture activity. The yellow dashed line represents the rough time point at which the assembly mechanism switches for a given row/reference sample. For sample comparisons in a single port, the RC_bray heatmap is symmetrical with respect to the main diagonal; therefore, only the upper triangle is displayed. (B) RC_bray values for cross-port sample comparisons between PI and PB. In this context, homogenizing dispersal (pink) indicates strong hydraulic connectivity between ports, consistent with the close proximity between PI and PB along the permeable fracture network. Boxes along the main diagonal (i.e., comparison between samples from different ports on the same day) have black boundaries for clarity. RC_bray values for the rest of the single-/cross-port pairwise comparisons not shown here are available in SI Appendix, Fig. S14.