(439d) 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.