BIODEGRADING BIOFILMS ON INNOVATIVE BIOPOLYMERIC SUPPORTS

ABSTRACT Water bioremediation is traditionally carried out using ‘ free ’ bacterial cells, however, in recent years, utilization of ‘immobilized’ bacterial cells on adsorbing matrices, has gained attention as a promising technique due to biotechnological and economic benefits (Sonawane et al., 2022). Bacterial biofilms show greater resilience, survival and degradative activity for longer periods than cells in the planktonic state (Alessandrello et al., 2017); moreover immobilization reduces bioremediation costs, eliminate cell dilution and dispersion in the environment (Bayat et al., 2015). Possible applications of immobilized biodegrading bacteria require long-term survival and maintenance of biodegrading performances. In this study, combinations of polylactic acid (PLA) and polycaprolactone (PCL) biodegradable membrane carriers hosting selected HC-biodegrading marine and soil bacterial biofilms were tested after different incubation periods and their survival was monitored over time, simulating storage effects. Results Soil hydrocarbon (HC) degrading actinobacteria and marine hydrocarbonoclastic bacteria were immobilized on absorbent biodegradable biopolymeric polylactic acid (PLA) and polycaprolactone (PCL) membranes (Scaffaro et al., 2017, Catania et al., 2020). Combinations of HC-degrading bacteria and biopolymers were obtained and tested on hexadecane. After 5, 10 and 15 days incubation, the capacity of adhesion and proliferation of bacterial cells into the biopolymers was verified by scanning electron microscopy (SEM); PLA and PCL nanofibers were covered by bacterial cells already after 5 days incubation; Total biomass (estimated as total dsDNA) extracted from biofilms confirmed the colonization up to 15 days incubation. Viable plate counts showed that survival of the bacterial strains was high for the entire experimental period. HC biodegradation ability of biofilms was assessed by high resolution GC-FID analysis, after extraction of total residual HC from the liquid medium and from biopolymers, incubated for different times. HC degradation was observed during the whole experiment and resulted higher in respect to the free-living bacterial cultures. Survival tests of bacterial biofilms adsorbed on biopolymers for up to 30 days are in progress. Conclusions The synergistic exploitation of the high absorbent capacity of biodegradable nanofiber membranes and the catabolic capacity of HC-degrading bacteria allow to obtain biodegrading biofilms endowed with higher removal capacity of hexadecane in respect to free-living bacterial cultures. The survival and biodegrading performances of the biofilm-carrier systems is maintained after 30 days incubation. A green, low-cost, biodegradable and reusable bioremediation tool is obtained without negative impacts on the environment. References: Alessandrello, M. J., Tomás, M. S. J., Raimondo, E. E., Vullo, D. L. and Ferrero, M. A. “Petroleum oil removal by immobilized bacterial cells on polyurethane foam under different temperature conditions”, Marine pollution bulletin, 122(1-2), 156-160 (2017). Bayat, Z., Hassanshahian, M. and Cappello, S. “Immobilization of microbes for bioremediation of crude oil polluted environments: a mini review”, The open microbiology journal, 9, 48 (2015). Catania, V., Lopresti, F., Cappello, S., Scaffaro, R. and Quatrini, P. “Innovative, ecofriendly biosorbent-biodegrading biofilms for bioremediation of oil-contaminated water”, New Biotechnology, 58, 25-31 (2020). Scaffaro, R., Lopresti, F., Catania, V., Santisi, S., Cappello, S., Botta, L. and Quatrini, P. “Polycaprolactone-based scaffold for oil-selective sorption and improvement of bacteria activity for bioremediation of polluted water: Porous PCL system obtained by leaching melt mixed PCL/PEG/NaCl composites: Oil uptake performance and bioremediation efficiency”, European Polymer Journal, 91, 260-273 (2017). Sonawane, J. M., Rai, A. K., Sharma, M., Tripathi, M. and Prasad, R. “Microbial biofilms: Recent advances and progress in environmental bioremediation”, Science of The Total Environment, 153843 (2022). Catania, V., Santisi, S., Signa, G., Vizzini, S., Mazzola, A., Cappello, S., ... & Quatrini, P. (2015). Intrinsic bioremediation potential of a chronically polluted marine coastal area. Marine Pollution Bulletin, 99(1-2), 138-149. Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A. M., & Quatrini, P. (2011). Involvement of an alkane hydroxylase system of Gordonia sp. strain SoCg in degradation of solid n-alkanes. Applied and environmental microbiology, 77(4), 1204-1213. Quatrini, P., Scaglione, G., De Pasquale, C., Riela, S., & Puglia, A. M. (2008). Isolation of Gram‐positive n‐alkane degraders from a hydrocarbon‐contaminated Mediterranean shoreline. Journal of applied microbiology, 104(1), 251-259