Changes in lipid and proteome composition accompany growth ofBacillus subterraneusMITOT1 under supercritical CO₂and may promote acclimation to associated stresses

Abstract

24Recent demonstration that multiple Bacillus strains grow in batch bioreactors containing 25 supercritical (sc) CO 2 (i.e. >73 atm, >31˚C) is surprising given the recognized roles of scCO 2 as 26 a sterilant and solvent. Growth under scCO 2 is of interest for biotechnological applications and 27 for microbially-enhanced geologic carbon sequestration. We hypothesize that Bacillus spp. may 28 alter cell wall and membrane composition in response to scCO 2 -associated stresses. In this study, 29 protein expression and membrane lipids of B. subterraneus MITOT1 were profiled in cultures 30 grown under headspaces of 1 and 100 atm of CO 2 or N 2 . Growth under 100 atm CO 2 revealed 31 significantly decreased fatty acid branching and increased fatty acyl chain lengths relative to 1 32 atm cultures. Proteomes of MITOT1 grown under 1 and 100 atm pressures of CO 2 and N 2 were 33 similar (Spearman R>0.65), and principal component analysis revealed variation by treatment 34 with the first two principal components corresponding to headspace gas (CO 2 or N 2 ) and pressure 35(1 atm and 100 atm), respectively. Amino acid metabolic proteins were enriched under CO 2 , 36 including the glycine cleavage system, previously shown to be upregulated in acid stress 37 response. These results provide insights into the stationary phase physiology of strains grown 38 under scCO 2 , suggesting modifications of cell membranes and amino acid metabolism may be 39 involved in response to acidic, high CO 2 conditions under scCO 2 . 40 41 42 43 Nevertheless, the cellular responses observed during these experiments suggest that alterations to 63 cell membranes and global changes to protein expression may be necessary to acclimate to 64 scCO 2 -assocated stresses. 65 4We have recently documented growth of Bacillus spp. under a scCO 2 headspace (3), 66 however how cells acclimate to grow under scCO 2 is unknown. Individual factors associated 67 with scCO 2 (e.g. elevated pressure, solvent, and acid stress) may have opposing influences on 68 cellular acclimation. High pressures compress membrane lipids with lipid disordering caused by 69 dissolution of gases into membrane bilayers (14). Bacteria under high pressure may compensate 70 by producing more unsaturated lipids or by decreasing lipid chain length in order to increase and 71 maintain membrane fluidity (15, 16), a response similar to that observed following exposure to 72 low temperature (16)(17)(18)(19)(20). However, solvent stress has been demonstrated to increase the 73 proportion of saturated fatty acid content and increasing acyl chain lengths (21). Bacteria also 74 alter membrane lipids in response to pH and oxygen availability. Acid stress in B. subtilis and 75Clostridium acetobutylicum was linked to a decreased proportion of branched and unsaturated 76 fatty acids, which was suggested to increase membrane rigidity and decrease the proton flux 77 across membranes (22, 23). Anaerobic growth in B. subtilis results in increased lipid chain length 78 (24) which may also promote membrane rigi...