While many physico-chemical approaches are available for degradation of per- and polyfluoroalkyl substances (PFASs), such as adsorption, photooxidation, and sonochemical pyrolysis, these options tend to be energy- and cost-intensive, which are impractical and expensive to be implemented in situ for the remediation of contamination. Other treatment methods, such as activated carbon adsorption and ion exchange resins, do not ensure detoxification of PFASs. While biological treatment using microbial whole cells is promising, it is constrained by geochemical characteristics and by concerns of biofouling, potentially releasing pathogens, and affecting microbial ecology of the natural and engineered systems. Using their extracellular enzymes directly was a promising alternative approach, because in vitro enzymes tend to be more efficient, and less constrained by the requirements for microbial growth. The limited stability of free enzymes in natural environments, however, restricts their implementation.

Phase I of this study proposed to develop an innovative in situ bioremediation technology using vault nanoparticles packaged with biodegradative enzymes that will facilitate the degradation of PFASs, and potentially other water contaminants. It was hypothesized that packaging active enzymes into vault nanoparticles for remediation of PFASs will eliminate the reliance on fungal growth and repeated injections of purified enzymes by enhancing in situ stability of multiple enzymes simultaneously. The study aimed to develop a single-step method for encapsulating ligninolytic enzymes (lignin peroxidase [LiP], manganese peroxidase [MnP], and laccase enzymes) in recombinant vaults. Phase I is complete and initial studies successfully confirmed that packaging fungal ligninolytic enzymes in vaults increased their stability as well as biotransformation rates of phenol, triclosan and BPA. Studies also successfully demonstrated packaging of MnP and LiP into vaults. However, due to limited enzyme concentration and incubation time, the results for PFAS transformation were not as significant. The results of Phase I studies can be found in the Phase I Final Report.

In Phase II, the focus will be on the degradation of perfluorooctanoic acid (PFOA) and fluorotelomer alcohols (FTOHs). Key objectives are to (1) Continue testing biotransformation of PFOA and 6:2 FTOH by MnP packaged in vaults; (2) To package laccase in vaults and compare the activity of the vault-packaged enzyme with the natural enzymes; and (3) To test the biotransformation of PFOA and 6:2 FTOH by vault-packaged laccase. The ultimate goal is to provide an innovative in situ remediation technology for biodegradation of select PFASs using ligninolytic enzymes, enhanced and protected by packaging in vault nanoparticles, thus eliminating the dependence on live organisms for biodegradation of the contaminants.

In Phase II, laboratory studies will be performed to produce recombinant laccase and MnP, each modified with an INT targeting domain to ensure high specificity in packaging. The enzyme activity assays for INT modified enzymes will be conducted over extended periods of time to quantify the stability and activity of the modified enzymes. The ability of these enzymes to biotransform a range of concentrations (10-1000 μg/L) of 6:2 FTOH and PFOA will be determined. Next, INT modified enzymes will be packaged into vaults and the activity and stability of vault-packaged enzymes will be quantified using enzyme activity assays and calibrated to that of purified free enzymes. Vault packaged MnP and laccase will then be evaluated for their ability to transform 6:2 FTOH and PFOA in the presence of various electron acceptors (02 and H202) and mediators (1-hydroxybenzotriazole and Suwanee River Natural Organic Matter). Biotransformation rates and half-saturation constants will be compared to those of purified enzymes and two wood-rotting fungi. The samples will be monitored for concentrations of PFASs and their metabolites, fluoride ions, and enzymatic activity.

Under Phase I of the study, INT-modified LiP and MnP were heterologously expressed in Sf9 insect cell lines intracellularly as nsLiP-INT and nsMnP-INT through Bac-to-Bac expression systems. Cell lysates containing nsLiP-INT or nsMnP-INT did not show significant peroxidase activities, suggesting that signal peptide processing contributes to correct folding of peroxidases and is likely required to activate these enzymes.

INT-fused MnP was also expressed extracellularly in Sf9 cell lines as sMnP-INT using its natural signal sequence. Vault-packaged sMnP-INT enzymes were isolated via ultracentrifuging mixtures of empty vault nanoparticles and culture supernatants containing sMnP-INT molecules. The integrity of vaults packaged with sMnP-INT was confirmed with negative stain transmission electron microscopy. Both free sMnP-INT and vault-packaged sMnP-INT showed manganese(II) ion dependent activity identical to that of MnP produced by P. chrysosporium, revealing that sMnP-INT maintained its activity when packaged inside vaults. Thermal stability of packaged sMnP-INT was compared with that of free sMnP-INT and MnP from P. chrysosporium, and it was found that vaults packaged sMnP-INT underwent slower deactivation at 20°C, 30°C and 40°C. Further deactivation kinetics study showed the enhanced thermal stability was due to the constraint from the vault shell, which prevents the enzymes from conformational changes. Empty vault nanoparticles showed excellent structural stability in the presence of PFCs in a phosphate buffer. Intact vault structure was maintained for at least 32 days. Additionally, to understand the performance of vault-packaged sMnP-INT under more realistic conditions, researchers examined the activity of sMnP-INT-vault and sMnP-INT under pH values ranging from 2.5 to 6.0 because MnP exhibits optimum activity at pH 4.0. The data showed sMnP-INT packaged in vaults had better activity at weakly acidic pH (5.0-6.0), indicating that vault nanoparticles were able to maintain the activity of packaged sMnP-INT against large pH changes.

INT-fused laccase was expressed as a secreted enzyme in Pichia pastoris under methanol inducing conditions, and was demonstrated to be active on methanol containing agar. However, the activity of recombinant laccase was below the detection limit when grown in liquid medium. Thus, it was not packaged into vaults in the duration of the one-year project.

Neither fungal whole cells of P. chrysosporium and T. versicolor, nor free enzymes including LiP, MnP, and laccase could transform PFOA under the limited experimental conditions used in this study. Free and vault packaged sMnP-INT enzymes were also tested, but no statistically significant transformation was observed. Possible reasons for little to no PFOA transformation include short incubation times, low enzyme concentrations, lack of mediators, and analytical limitations.

This project will support SERDP's mission to reduce the DoD's liabilities by developing sustainable, cost-effective technologies for expedited site cleanup and closure by in situ remediation of PFAS contamination in soil, sediments, and water. While PFOA and perfluorooctane sulfonate (PFOS) are important contaminants of concern for the DoD, biotransformation of their precursors, such as FTOHs, in natural or engineered processes via pathways that do not form terminal compounds would be valuable for reducing the overall environmental burden of PFASs. These potential advantages of utilizing vaults packaged enzymes over free enzymes and live microbes will enable the widespread application of this technology to a large number of DoD sites. The integration of two novel technologies, enzymatic bioremediation with vaults packaging to degrade PFASs will be a transformative step towards in situ bioremediation of these contaminants with the potential to lead to more customized enzyme catalyzed solutions for hazardous waste treatment and contaminated sites.

Merino, N., Y. Qu, R.A. Deeb, E.L. Hawley, M.R. Hoffmann, and S. Mahendra. 2016. Degradation and Removal Methods for Perfluoroalkyl and Polyfluoroalkyl Substances in Water. Environmental Engineering Science, 33(9):615-649.

Wang, M., D. Abad, V. A. Kickhoefer, L. H. Rome, and S. Mahendra. 2018.  Encapsulation of Exogenous Proteins in Vault Nanoparticles. Protein Scaffolds: Design, Synthesis, and Applications, Humana Press. 25-37.

Wang, M., V. A. Kickhoefer, E. H. Lan, B. S. Dunn, L. H. Rome, and S. Mahendra. 2021. Vault Nanocapsule-Mediated Biomimetic Silicification for Efficient and Robust Immobilization of Proteins in Silica Composites. Chemical Engineering Journal, 418:129406.

Wang, M., Y. Chen, D. Abad, V. A. Kickhoefer, P. Allard, L. H. Rome, and S. Mahendra. 2019.  A Vault-Encapsulated Enzyme Approach for Efficient Degradation and Detoxification of Bisphenol A and its Analogues.  ACS Sustainable Chemistry and Engineering, 7(6):5808-5817.

Zhang, L., N. W. Johnson, Y. Liu, Y. Miao, R. Chen, H. Chen, Q. Jiang, Z. Li, Y. Dong, and S. Mahendra. 2020. Biodegradation Mechanisms of Sulfonamides by Phanerochaete chrysosporium-Luffa Fiber System Revealed at the Transcriptome Level. Chemosphere, 266:129194.

Kalra, S. S. 2021. Ultrasound and Fungi Mediated Remediation of Model Emerging Contaminants: Per- and Polyfluoroalkyl Substances & Nitrotriazolone. (Ph.D. Thesis). University of California, Los Angeles.

Wang, M. 2018. Vault Nanoparticles-Based Enzyme Immobilization Platform for Environmental Applications. (Ph.D. Thesis). University of California, Los Angeles.