Abstract
The phospholipid fatty acid (PLFA) content was determined in samples of Paracoccus denitrificans encapsulated in silica hydrogel films prepared from prepolymerized tetramethoxysilane (TMOS). Immediately after encapsulation the total PLFA concentration was linearly proportional to the optical density (600 nm) of the input microbial suspension (R2 = 0.99). After 7 days this relationship remained linear, but with significantly decreased slope, indicating a higher extinction of bacteria in suspensions of input concentration 108 cells/mL and higher. trans-Fatty acids, indicators of cytoplasmatic membrane disturbances, were below the detection limit. The cy/pre ratio (i.e., ratio of cyclopropylated fatty acids (cy17:0 + cy19:0) to their metabolic precursors (16:1ω7 + 18:1ω7)), an indicator of the transition of the culture to a stationary growth-phase, decreased depending on co-immobilization of nutrients in the order phosphate buffer > mineral medium > Luria Broth rich medium. The ratio, too, was logarithmically proportional to cell concentration. These results confirm the applicability of total PLFA as an indicator for the determination of living biomass and cy/pre ratio for determination of nutrient limitation of microorganisms encapsulated in sol-gel matrices. This may be of interest for monitoring of sol-gel encapsulated bacteria proposed as optical recognition elements in biosensor construction, as well as other biotechnological applications.
Highlights
Optimized biocompatible sol-gel routes have recently enabled encapsulation of living cells into inorganic hydrogels forming biocomposite materials with interesting application properties [1,2,3,4].Silica matrices, due to their transparency, are especially useful for preparation of recognition elements of optical biosensors, utilizing encapsulated bioluminescent or fluorescent bioreporters [5,6,7,8].Despite optimization, different stresses are imposed on microorganisms both during the encapsulation procedure as well as in the final encapsulated state [1,9]
In this study we aimed to test the applicability of this approach to microorganisms encapsulated in rigid silica matrix prepared by sol-gel method and evaluate a few possible stress indicators in response to nutrient insufficiency
Medium and concentrations of total phospholipid fatty acid (PLFA) and several abundant fatty acids methyl esters (FAME) one day after encapsulation
Summary
Optimized biocompatible sol-gel routes have recently enabled encapsulation of living cells into inorganic hydrogels forming biocomposite materials with interesting application properties [1,2,3,4].Silica matrices, due to their transparency, are especially useful for preparation of recognition elements of optical biosensors, utilizing encapsulated bioluminescent or fluorescent bioreporters [5,6,7,8].Despite optimization, different stresses are imposed on microorganisms both during the encapsulation procedure as well as in the final encapsulated state [1,9]. Viability and stress of encapsulated microorganisms have been studied using many ways such as colony-forming units, various microscopy techniques, fluorescence, bioluminescence, metabolic and enzyme activities, or gene expression (see review [1] for details). We have demonstrated the usefulness of PLFA determination for characterization of viability of encapsulated microorganisms namely in polyvinyl alcohol (PVA) during biotechnological processes [15]. This preliminary study was focused on total PLFA indicator only. In this study we aimed to test the applicability of this approach to microorganisms encapsulated in rigid silica matrix prepared by sol-gel method and evaluate a few possible stress indicators (ratios of trans/cis PLFA and cy/pre PLFA) in response to nutrient insufficiency
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