(601d) Development and Optimization of Poly(vinyl) Alcohol-Alginate Hydrogel Beads for Immobilization of Rhodococcus Rhodochrous ATCC 21198 and Slow-Release Compounds

Introduction: Groundwater contamination from chlorinated aliphatic hydrocarbons (CAHs) such as cis-1,2-dichloroethene (cDCE) is a widespread issue in the United States. Passive, long-term bioremediation with bacteria immobilized in hydrogels is a promising remediation technique. Rhodococcus rhodochrous ATCC 21198 is an alkane-oxidizing aerobic bacterium that degrades cDCE through cometabolism, where contaminants are transformed but not utilized as a carbon or energy source. Our initial studies immobilized ATCC 21198 in a gellan gum (GG) hydrogel bead with a slow-release compound, tetrabutoxysilane (TBOS) that hydrolyzes over time into a carbon and energy source that can be utilized by ATCC 21198. GG beads rapidly degraded, making them unfit for long term ground water remediation applications. Poly(vinyl) alcohol (PVA) and Alginate (Alg) beads are durable, biodegradable beads that have been used to immobilize other bacteria strains. Thus, our objective was to engineer a hydrogel with increased compressive moduli and decreased hydrolysis rates.

Materials and Methods: Design of experiments, specifically a central composite orthogonal (CCO) design, was used to identify the cross linking time and concentrations of PVA and Alg that maximized the Young’s modulus and minimized the oxygen consumption rates at days 1 and 30. ATCC 21198 and TBOS were immobilized in PVA-Alg beads with t_crosslink = [30 - 120] min, C_PVA = [1 - 3] % (w/v), and C_Alg = [1 - 2] % (w/v). Beads were formed by dropping polymer solutions through a syringe needle into a crosslinking solution of 3 % (w/v) boric acid and 1.5 % (w/v) CaCl₂. Beads were placed into batch reactors and spiked with 250 ppb cDCE at days 1 and 30. cDCE and oxygen were measured with gas chromatography and rates were determined using zero-order kinetic models. Compression tests were performed to calculate the Young’s Modulus, E.

Results and Discussion: Overall, the Young’s moduli decreased (Fig 1.A) and the cDCE consumption rates increased after 30 days (Fig 1.B) due to cell growth in hydrogel beads. Oxygen rates also decreased (Fig 1.C), indicating high initial hydrolysis rates of TBOS. We successfully fit predictive models of Young’s modulus and hydrolysis as functions of crosslinking time and polymer concentrations and found that all three input variables had significant interactions with each other as well as significant effects on the output variables. These models were then used to identify the bead formulation that maximized stiffness and minimized hydrolysis rates.

Conclusion and Future Directions: DOE has been employed to identify the optimum hydrogel properties to promote long-term stability of hydrogel beads for bioremediation via immobilized cells. Future studies will focus on the polymer chain entanglement density and molecular weight of PVA, and cell growth based on the concentration and type of SRC.