Changes in environmental and engineered conditions alter the plasma membrane lipidome of fractured shale bacteria.

IMPORTANCE: Microorganisms inadvertently introduced into the shale reservoir during fracturing face multiple stressors including brine-level salinities and starvation. However, some anaerobic halotolerant bacteria adapt and persist for long periods of time. They produce hydrogen sulfide, which sours the reservoir and corrodes engineering infrastructure. In addition, they form biofilms on rock matrices, which decrease shale permeability and clog fracture networks. These reduce well productivity and increase extraction costs. Under stress, microbes remodel their plasma membrane to optimize its roles in protection and mediating cellular processes such as signaling, transport, and energy metabolism. Hence, by observing changes in the membrane lipidome of model shale bacteria, Halanaerobium congolense WG10, and mixed consortia enriched from produced fluids under varying subsurface conditions and growth modes, we provide insight that advances our knowledge of the fractured shale biosystem. We also offer data-driven recommendations for improving biocontrol efficacy and the efficiency of energy recovery from unconventional formations.

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

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.

Enhancement of the Anti-inflammatory Effect of Bromelain by Its Immobilization on Probiotic Spore of Bacillus cereus.

The therapeutic application of bromelain is limited due to its sensitivity to operating conditions such as high acidity, gastric proteases in the stomach juice, chemicals, organic solvents and elevated temperature. We hypothesized that bromelain immobilized on probiotic bacterial spores would show enhanced therapeutic activity through possible synergistic or additive effects. In this study, the oedema inhibition potential of bromelain immobilized on probiotic Bacillus spores was compared to the free enzyme using the carrageenan paw oedema model with Wistar rats. In batch A rats (carrageenan-induced inflammation 30 min after receiving oral treatments), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed the highest oedema inhibition, 89.20 ± 15.30%, while group 4 treated with a lower dose of free bromelain had oedema inhibition of 60.25 ± 13.00%. For batch B rats (carrageenan-induced inflammation after receiving oral treatment for three days), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed higher inhibition percentage (81.94 ± 8.86) than group 4 treated with a lower dose of free bromelain (78.45 ± 4.46) after 24 h. Our results showed that used alone, the enzyme and the spores produced oedema inhibition and improved the motility of the rats. The spore-immobilized bromelain formulation performed approximately 0.9-fold better than the free bromelain and the free spores at the lower evaluated dose.

Stabilizing enzymes by immobilization on bacterial spores: A review of literature.

The ever-increasing applications of enzymes are limited by the relatively poor performance in harsh processing conditions. As a result, there are constant innovations in immobilization protocols for improving biocatalyst activity and stability. Bacterial spores are cheap to generate and highly resistant to environmental stress. The spore core is sheathed by an inner membrane, the germ cell wall, the cortex, outer membrane, spore coat and in some species the exosporium. The spore surface is anion-rich, hydrophobic and contains several reactive groups capable of interacting and stabilizing enzyme molecules through electrostatic forces, hydrophobic interactions and covalent bonding. The probiotic nature of spores obtained from non-toxic bacterial species makes them suitable carriers for the enzyme immobilization, especially food-grade enzymes or those intended for therapeutic use. Immobilization on spores is by direct adsorption, covalent attachment or surface display during the sporulation phase. Hindrances to the immobilization on spore matrix include the production rates, operational instability, and reduced catalytic properties due to conformational changes in enzyme. This paper reviews bacterial spore as a heterofunctional support matrix gives reasons why probiotic bacillus spores are better options and the diverse technologies adopted for spore-enzyme immobilization. It further suggests directions for future use and discusses the commercialization prospects.

Stabilizing bromelain for therapeutic applications by adsorption immobilization on spores of probiotic Bacillus.

Bromelain, a protease from pineapple plant can be applied as oral drug for the treatment of inflammation and certain diseases. Unlike most conventional supports, immobilization on edible support will make the enzyme suitable for therapeutic use. In this study, spores of probiotic Bacillus sp was used for the adsorption of bromelain. Effect of pH, temperature and enzyme concentration on bromelain immobilization was studied, followed by characterization of the enzymes. Maximum bromelain coupling (%) (50.607 ±â€¯4.194) was obtained when immobilization was carried out at pH 6.0, 24 °C for 150 min. The immobilized enzyme showed optimum activity at pH 8 and 80 °C, while the free enzyme had 6 and 60 °C as its optimum pH and temperature, respectively. Bromelain Vmax increased after immobilization while Km decreased. Activation energy, Ea was 26.513 kJ/mol and 20.942 kJ/mol for the free and immobilized enzymes, respectively. The immobilized bromelain also showed significantly greater storage and thermal stability than the free bromelain. At 80 °C, the free bromelain lost all its activity after 50 min while the immobilized enzyme lost only 46.89% activity. Bromelain was successfully immobilized on Bacillus spores with improved catalytic and non-catalytic properties and this holds great potential for its growing therapeutic applications.