Bacterial Necromass Is Rapidly Metabolized by Heterotrophic Bacteria and Supports Multiple Trophic Levels of the Groundwater Microbiome

Abstract

Pristine groundwater is a highly stable environment with microbes adapted to dark, oligotrophic conditions. Input events like heavy rainfalls can introduce the excess particulate organic matter, including surface-derived microorganisms, thereby disturbing the groundwater microbiome. Some surface-derived bacteria will not survive this translocation, leading to an input of necromass to the groundwater. Here, we investigated the effects of necromass addition to the microbial community in fractured bedrock groundwater, using groundwater mesocosms as model systems. We followed the uptake of ¹³C-labeled necromass by the bacterial and eukaryotic groundwater community quantitatively and over time using a complementary protein-stable and DNA-stable isotope probing approach. Necromass was rapidly depleted in the mesocosms within 4 days, accompanied by a strong decrease in Shannon diversity and a 10-fold increase in bacterial 16S rRNA gene copy numbers. Species of Flavobacterium, Massilia, Rheinheimera, Rhodoferax, and Undibacterium dominated the microbial community within 2 days and were identified as key players in necromass degradation, based on a ¹³C incorporation of >90% in their peptides. Their proteomes comprised various proteins for uptake and transport functions and amino acid metabolization. After 4 and 8 days, the autotrophic and mixotrophic taxa Nitrosomonas, Limnohabitans, Paucibacter, and Acidovorax increased in abundance with a ¹³C incorporation between 0.5% and 23%. Likewise, eukaryotes assimilated necromass-derived carbon either directly or indirectly. Our data point toward a fast and exclusive uptake of labeled necromass by a few specialists followed by a concerted action of groundwater microorganisms, including autotrophs presumably fueled by released, reduced nitrogen and sulfur compounds generated during necromass degradation. IMPORTANCE Subsurface microbiomes provide essential ecosystem services, like the generation of drinking water. These ecosystems are devoid of light-driven primary production, and microbial life is adapted to the resulting oligotrophic conditions. Modern groundwater is most vulnerable to anthropogenic and climatic impacts. Heavy rainfalls, which will increase with climate change, can result in high surface inputs into shallow aquifers by percolation or lateral flow. These inputs include terrestrial organic matter and surface-derived microbes that are not all capable to flourish in aquatic subsurface habitats. Here, we investigated the response of groundwater mesocosms to the addition of bacterial necromass, simulating event-driven surface input. We found that the groundwater microbiome responds with a rapid bloom of only a few primary degraders, followed by the activation of typical groundwater autotrophs and mixotrophs, as well as eukaryotes. Our results suggest that this multiphase strategy is essential to maintain the balance of the groundwater microbiome to provide ecosystem services.

Keywords: AquaDiva; groundwater; metaproteomics; necromass; stable isotope probing; subsurface; surface input.

FIG 1

Abundances of Pseudomonas on DNA and peptide level. (A) Phylogenetic tree based on 16S rRNA gene sequences of the most abundant Pseudomonas OTUs as well the isolate Hainich_002 that was used for necromass generation (star symbol). The tree was calculated using the arb neighbor-joining method (1000 bootstraps) within arb (81, 82). Closely related Pseudomonas species were added as references. (B) Relative abundances of Pseudomonas-related OTUs increase after necromass was added on day 0 and then progressively decrease throughout the incubation. Likely, also members of the genus Pseudomonas partake in the initial degradation of Pseudomonas-derived necromass. OTU00019, the most closely related OTU to the isolate Hainich_002 is highlighted in yellow. (C) Number of peptides associated with the genus Pseudomonas within the original groundwater at day 0, as well as the ¹²C labeled incubations over time. Unlike at the DNA level, most Pseudomonas-related peptides presumably stem from the added necromass.

FIG 2

Bacterial abundances in groundwater and mesocosms supplemented with necromass. Average 16S rRNA gene copy numbers within the original groundwater (white), as well as within the incubations on days 2, 4, and 8 after necromass addition in the ¹²C (green) and ¹³C (orange) mesocosms. Error bars show the standard deviation of three samples. P values were derived from Student's t test and indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

Phylogenetic profiles based on relative abundances of bacterial 16S rRNA gene amplicons. Average relative abundances of bacterial families in the original groundwater, as well as the ¹²C (green) and ¹³C (orange) mesocosms over the 8 days of incubation. Shannon diversity indices are given as average values per time point.

FIG 4

Relative isotopic abundances (RIA) and the number of peptides in the most abundant genera. Boxplots show the uptake of labeled carbon by potential heterotrophic (dark green), autotrophic (yellow), and mixotrophic (blue) bacteria in the ¹³C incubations. The corresponding bar charts represent the numbers of identified peptides in the ¹²C incubations for the respective genus.

FIG 5

Functional classification (KEGG orthology) of identified peptides from the most abundant genera. Heterotrophic genera (dark green) show high abundances of peptides associated with the degradation and transport of amino acids as well as the other transporting mechanisms potentially involved in the uptake and breakdown of necromass. Potentially autotrophic (yellow) and mixotrophic (blue) bacteria show a less distinct pattern. For Nitrosomonas peptides from the key enzyme of carbon fixation via the CBB cycle, as well as enzymes participating in nitrogen metabolism were identified. Circle sizes indicate the number of identified peptides.

FIG 6

Evidence of necromass uptake by Eukaryotes via DNA-SIP. Relative abundances of eukaryotic 18S rRNA genes in the heavy and light DNA fractions after 8 days of incubation show a significant shift toward the heavy DNA fractions in the incubations with ¹³C-labeled necromass (P < 0.001; Fisher’s exact test; Table S5), indicating that eukaryotes have been taking up necromass derived ¹³C during the experiment.

FIG 7

Conceptual view of the response of the groundwater microbiome to a disturbance by large inputs of necromass derived carbon. ¹³C labeled necromass (orange) is taken up by heterotrophic (green) members of the community that can rapidly thrive on the added OC. By metabolizing, e.g., amino acids contained in necromass, excess nitrogen, and sulfur compounds are being released and can subsequently stimulate the growth of autotrophic (yellow) and mixotrophic (blue) bacteria. Simultaneously, eukaryotes (purple) are taking up necromass-derived ¹³C by feeding on the heterotrophic community members or necromass directly.