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This publication describes in detail the immobilization of Micrococcus luteus (M. luteus) in various hydrogels and the following processing into water-stable living composites.
One part of the publication deals with the immobilization of bacteria into poly(vinyl alcohol) (PVA) microparticles with a diameter of about 5 µm. The bacteria are entrapped by utilizing an aqueous solution of PVA and a high velocity stirrer. Afterwards, the particles are formed through the addition of acetone. The immobilized bacteria survived the storage in various organic solvents and endured chemical surface reaction under different conditions, as shown by using hexamethylene diisocyanate (HDMI), toluene diisocyanate (TDI) and octadecyl isocyanat. After the surface reactions, living immobilized bacteria were proven by inoculating and incubating agar plates containing nutrient.
One major drawback of the living composite is its behavior in presence of water. The PVA-particles swell and hence the bacteria are released into the surrounding medium. We have overcome this disadvantage by polymerizing various amounts of poly(methyl methacrylate) (PMMA) onto the PVA-particles in presence of the living bacteria inside the particles. In a first step, we introduced an ATRP-initiator by a chemical surface reaction between the OH-groups and the acid bromid of the initiator. In a second step, the grafting from polymerization of methyl methacrylate (MMA) was carried out, leading to exactly one PMMA shell around each PVA particle. We were able to control the amount of the initiator as well as the amount of the PMMA. The core-shell structure was proven by utilizing TEM and confocal microscopy. Furthermore, we demonstrated the hydrophobic behavior of the core-shell particles in terms of the release of immobilized fluorescein. In addition, the retardation of the release of bacteria was verified by inoculating agar plates containing nutrition and buffer solution. The water stability of the living composite was shown by using SEM and Cryo-SEM.
As a result, we received water-stable PMMA-PVA-core-shell particles with immobilized living bacteria. We also proved the survival of the bacteria during the complete process.
Moreover, the free bacteria were immobilized in nano- and microfibers consisting of poly(ethylene oxide) (PEO) and PVA. The nanofibers were produced through the electrospinning technique whereas the microfibers were created by wet spinning. The hydrogel fibers were coated with poly(p-xylylene) (PPX) via chemical vapor deposition (CVD). The bacteria hence were immobilized in core-shell fibers with an adjustable thickness. The fibers were water-stable and prevented the release of bacteria into the surrounding medium. The bacteria survived the process and we demonstrated their metabolic activity by the reduction of resazurin. Moreover, we utilized the living fiber composites for the sequestration of gold ions from aqueous solution. The decrease of the amount of gold in the solution was demonstrated by ICP-MS. The deposition of gold inside the fibers was verified by TEM and EDX.
Concluding, we were able to immobilize bacteria in hydrogel particles and hydrogel fibers. Furthermore, we proved the feasibility of chemical surface reactions for creating water-stable living composites. The immobilized bacteria survived these reactions. Moreover, we demonstrated their metabolic activity as well as their ability to sequestrate heavy metal ions from aqueous solutions. The water-stable composites are of interest for the bioremediation of polluted water and for biotransformation.