265715 Engineering Biomaterials for Bioremediation Applications
To be useful in a water-treatment applications, a bioremediation method should: (i) have stable, long-term degradation activity; (ii) be mechanically stable and sturdy; (iii) be conducive to high water flow; (iv) maintain active enzymes or cells in the matrix without significant release, and (v) be inexpensive. We developed hybrid biomaterials to be used for atrazine (2-chloro-4-ethylamine-6-isopropylamino-s-triazine) biodegradation. The herbicide atrazine is currently used in 70 countries at an estimated annual rate of 111,000 tons for control of broadleaf weeds, principally in corn, sorghum, and sugarcane. The hybrid biomaterial consisted of recombinant E. coli cells overexpressing atrazine chlorohydrolase (AtzA), which converts atrazine into hydroxyatrazine. The cells were encapsulated in a polymer/silicon oxide matrix by a sol-gel process (Figure 1).
Figure 1: SEM image of E-coli encapsulated in silica gels
The silica base gel used to encapsulate the microorganisms consisted of a combination of silicon oxide precursors (e.g., silica nanoparticles, alkoxides) and a biocompatible organic polymer (e.g., polyethylene glycol, PEG). The porous material enabled diffusion of water and atrazine into the gel and diffusion of hydroxyatrazine out of the gel. The gel additionally adsorbed atrazine, a property that contributed to removal of atrazine from the solution in the process. Moreover, high temperature exposure was utilized to ensure that the encapsulated cells were non-viable but remained fully active in degrading atrazine over a long time scale.
Figure 2: Specific activity of encapsulated (vs. free) cells measured over 4 months.
We measured the activities of the free and silica encapsulated cells over 4 months (Figure 2). When the activity was measured at room temperature, free cells showed an average of (0.61 ± 0.04) mmol/g/min of activity over 21 days. After 21 days, significant cell lysis was observed in the free cells; this was likely due to long-term hypo-osmotic stress induced by water. Therefore, the experiments on the free cells were stopped at that time point. On the other hand, cells encapsulated in porous silica gels showed stable activity between (0.44 ± 0.06) mmol/g/min to (0.66 ± 0.12) mmol/g/min for up to 4 months. This showed that even though the encapsulated cells were non-viable and had lost some membrane integrity, AtzA was protected and active in the silica matrix. The activities of the free and encapsulated cells were found to be temperature dependent. At 4°C, activity dropped by 45% and 30% for the free and encapsulated cells, respectively. The activity of encapsulated cells at 4°C was 33.3% higher than the cells in solution.
This showed that bioremediation using encapsulated microorganisms is a safe and feasible technique. Currently, we are working towards using similar techniques to remove chemicals from wastewaters generated by hydraulic fracturing for shale gas and oil.
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