Aromatic amino acid metabolism and active transport regulation are implicated in microbial persistence in fractured shale reservoirs

Abstract

Hydraulic fracturing has unlocked vast amounts of hydrocarbons trapped within unconventional shale formations. This large-scale engineering approach inadvertently introduces microorganisms into the hydrocarbon reservoir, allowing them to inhabit a new physical space and thrive in the unique biogeochemical resources present in the environment. Advancing our fundamental understanding of microbial growth and physiology in this extreme subsurface environment is critical to improving biofouling control efficacy and maximizing opportunities for beneficial natural resource exploitation. Here, we used metaproteomics and exometabolomics to investigate the biochemical mechanisms underpinning the adaptation of model bacterium Halanaerobium congolense WG10 and mixed microbial consortia enriched from shale-produced fluids to hypersalinity and very low reservoir flow rates (metabolic stress). We also queried the metabolic foundation for biofilm formation in this system, a major impediment to subsurface energy exploration. For the first time, we report that H. congolense WG10 accumulates tyrosine for osmoprotection, an indication of the flexible robustness of stress tolerance that enables its long-term persistence in fractured shale environments. We also identified aromatic amino acid synthesis and cell wall maintenance as critical to biofilm formation. Finally, regulation of transmembrane transport is key to metabolic stress adaptation in shale bacteria under very low well flow rates. These results provide unique insights that enable better management of hydraulically fractured shale systems, for more efficient and sustainable energy extraction.

Keywords: Halanaerobiumfractured shale; hydraulic retention time; metabolomics; produced water; proteomics; salinity.

Figure 1

Protein and exometabolome changes in H. congolense WG10 grown under 13% (optimum) vs. 20% (high) NaCl. (A) Volcano plot showing the P-value and log2 FC of proteins detected in H. congolense WG10 planktonic cells grown at 24 h HRT under 13% vs. 20% NaCl (N = 3). The direction of comparison is 13% to 20%. The dotted horizontal line indicates the threshold of statistical significance, P < .05, while both dotted vertical lines delineate |FC| > 1.5. (B) Functional enrichment of discriminant (FC > 1 and P < .05) proteins in H. congolense WG10 planktonic cells upregulated under 20% NaCl. Bubble size indicates gene count. GO BP, gene ontology biological process; GO CC, gene ontology cellular compartment; GO MF, gene ontology molecular function; PIR SF, protein information resource superfamily; UP KW BP, UniProt keywords biological process; UP KW CC, UniProt keywords cellular compartment; UP SEQ, UniProt sequences. (C) Heatmap of normalized and scaled concentrations of extracellular metabolites produced by H. congolense WG10 planktonic cells grown at 24 h HRT, under 13% vs. 20% NaCl, annotated with a bar plot showing the log2 FC and corresponding P-values (N = 3). Statistical significance is defined as |FC| > 1.5 and P < .05. Rows and columns are clustered by Pearson’s correlation.

Figure 2

Sodium-proton antiporter and tyrosine biosynthesis are upregulated in H. congolense WG10 growing under hypersalinity. Schematic of reaction steps in tyrosine biosynthesis upregulated in H. congolense WG10 growing under high (20% NaCl) salinity relative to the optimum (13%). Significantly (P < .05) upregulated proteins (enzymes) are bolded and italicized. Triangles and diamonds indicate upregulation in planktonic and biofilm cultures, respectively. Solid shape line denotes FC > 1.0 while dotted shape line denotes 0 < FC < 1.0. PFK, phosphofructokinase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGAM, phosphoglycerate mutase; DAH7PS, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase; CS, chorismate synthase; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; HPA, histidinol-phosphate aminotransferase; TYRA, arogenate dehydrogenase.

Figure 3

Proteomic changes occurring in H. congolense WG10 and PFE mixed microbial consortia during biofilm formation. (A) Volcano plot showing the P-value and log2 FC of proteins in H. congolense WG10 growing under 13% NaCl in planktonic (HRT: 48 h) vs. biofilm mode (48 h incubation). The direction of comparison is planktonic to biofilm. (B) Volcano plot showing the P-value and log2 FC of proteins in mixed microbial consortia enriched from shale-produced fluids in planktonic (HRT: 48 h) vs. biofilm mode. The direction of comparison is planktonic to biofilm. In the volcano plots, the horizontal line indicates the threshold of statistical significance, P < .05, while both vertical lines delineate |FC| > 1.5. (C) Functional enrichment of proteins that significantly increased (FC > 1; P < .05) in H. congolense WG10 during biofilm formation. Bubble size indicates gene count. GO BP, gene ontology biological process; GO CC, gene ontology cellular compartment; GO MF, gene ontology molecular function; PIR SF, protein information resource superfamily; UP KW BP, UniProt keywords biological process; UP KW CC, UniProt keywords cellular compartment; UP KW MF, UniProt keywords molecular function.

Figure 4

Proteins catalyzing key steps in cell wall and lipopolysaccharide biosynthesis are significantly (P < .05) upregulated in H. congolense WG10 biofilms relative to planktonic (HRT: 48 h) cells, both of which were incubated under optimal salinity (13% NaCl). Upregulation is denoted by upward-facing triangles. Dotted lines indicate multiple reaction steps. DapA, 4-hydroxy-tetrahydropicolinate synthase; GTs, glycosyltransferases; DacA, D-alanyl-D-alanine carboxypeptidase; LpxC, UDP-3-0-acyl-N-acetylglucosamine deacetylase.

Figure 5

Proteomic changes in H. congolense WG10 and PFE consortia grown under high HRT (48 h) vs. low (19.2 h). (A, B) Volcano plots showing the P-value and log2 FC of proteins in H. congolense WG10 (A) and PFE (B) planktonic cultures, grown under 19.2 h vs. 48 h HRT. The direction of comparison is 19.2 to 48 h. Halanaerobium congolense cultures were incubated at optimal salinity (13% NaCl). The horizontal line indicates the threshold of statistical significance, P < 0.05, while both vertical lines delineate |FC| > 1.5. (C, D) Functional enrichment of proteins that significantly increased (FC > 1; P < .05) in H. congolense WG10 (C) and PFE microbial consortia (D), during growth under 48 h HRT relative to 19.2 h. Bubble size indicates gene count. GO BP, gene ontology biological process; GO CC, gene ontology cellular compartment; GO MF, gene ontology molecular function; PIR SF, protein information resource superfamily; UP KW BP, UniProt keywords biological process; UP KW CC, UniProt keywords cellular compartment; UP KW MF, UniProt keywords molecular function; UP SEQ, UniProt sequences.

Figure 6

Proposed model for key physiological mechanisms underlying microbial persistence in fractured shale. (A) Hypersalinity tolerance mechanisms include de novo AAAs (tyrosine) accumulation, and uptake of salts (salt-in) and osmolytes (compatible solutes) from the cell’s surroundings. (B) Increase in AAA synthesis and cell envelope maintenance are vital for biofilm formation. (C) Facing metabolic stress, cells ramp up amino acid/protein metabolism and express more RND transporters to extrude toxic metabolic waste.