Salinity and hydraulic retention time induce membrane phospholipid acyl chain remodeling in Halanaerobium congolense WG10 and mixed cultures from hydraulically fractured shale wells

Abstract

Bacteria remodel their plasma membrane lipidome to maintain key biophysical attributes in response to ecological disturbances. For Halanaerobium and other anaerobic halotolerant taxa that persist in hydraulically fractured deep subsurface shale reservoirs, salinity, and hydraulic retention time (HRT) are important perturbants of cell membrane structure, yet their effects remain poorly understood. Membrane-linked activities underlie in situ microbial growth kinetics and physiologies which drive biogeochemical reactions in engineered subsurface systems. Hence, we used gas chromatography-mass spectrometry (GC-MS) to investigate the effects of salinity and HRT on the phospholipid fatty acid composition of H. congolense WG10 and mixed enrichment cultures from hydraulically fractured shale wells. We also coupled acyl chain remodeling to membrane mechanics by measuring bilayer elasticity using atomic force microscopy (AFM). For these experiments, cultures were grown in a chemostat vessel operated in continuous flow mode under strict anoxia and constant stirring. Our findings show that salinity and HRT induce significant changes in membrane fatty acid chemistry of H. congolense WG10 in distinct and complementary ways. Notably, under nonoptimal salt concentrations (7% and 20% NaCl), H. congolense WG10 elevates the portion of polyunsaturated fatty acids (PUFAs) in its membrane, and this results in an apparent increase in fluidity (homeoviscous adaptation principle) and thickness. Double bond index (DBI) and mean chain length (MCL) were used as proxies for membrane fluidity and thickness, respectively. These results provide new insight into our understanding of how environmental and engineered factors might disrupt the physical and biogeochemical equilibria of fractured shale by inducing physiologically relevant changes in the membrane fatty acid chemistry of persistent microbial taxa. GRAPHICAL ABSTRACTSalinity significantly alters membrane bilayer fluidity and thickness in Halanaerobium congolense WG10.

Keywords: Halanaerobium; fatty acid methyl ester; fractured shale; hydraulic retention time; lipids; membrane adaptation; salinity.

GRAPHICAL ABSTRACT

Salinity significantly alters membrane bilayer fluidity and thickness in Halanaerobium congolense WG10.

Figure 1

Heatmap distribution of individual phospholipid fatty acids in Halanaerobium congolense WG10 and fluid enrichment (mixed) cultures grown at 40°C under different salinities and HRTs. Fatty acids are sorted by class, then chain length and number of double bonds.

Figure 2

Membrane-derived fatty acids that significantly varied with salinity in H. congolense WG10. (A) 14:1ω5; (B) 15:1ω5c; (C) 16:1ω9c; (D) 17:0; (E) 18:0; (F) 18:1ω7c; (G) 18:2ω4; (H) 20:4ω6; (I) 22:4ω6c. Fatty acids are arranged by chain length then by number of double bonds. p-Values are obtained from one-way ANOVA of independent groups. Variation is considered significant at α ≤ 0.05.

Figure 3

Mean chain length (MCL) and double bond index (DBI) of membrane phospholipids in H. congolense WG10 responded distinctly to salinity (A,B) and hydraulic retention time (HRT). The effect of HRT was evaluated at two discrete salinities – 7% (C,D) and 13% (E,F). p-Values are obtained from one-way ANOVA of multiple independent groups or Student’s t-test of two independent groups. Variation is considered statistically significant at α ≤ 0.05.

Figure 4

Variations in molar abundances of membrane phospholipid fatty acid classes in H. congolense WG10 with salinity at constant temperature (40°C) and HRT (24 h). p-Values are obtained from one-way ANOVA of independent groups. Variation is considered significant at α ≤ 0.05. SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid; BCFA, Branched chain fatty acid; CyclicFA, Cyclic fatty acid.

Figure 5

Variations in abundances of membrane PLFA classes in H. congolense WG10 with hydraulic retention time at constant temperature (40°C), evaluated at two different salinities – 7% (A–C) and 13% (D–F). p-Values are obtained from student’s t-test of independent groups. Variation is considered significant at α ≤ 0.05. SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid.