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- Optimizing bacterial nanocellulose production from eucalyptus bark: A circular approach to wastewater management and resource recoveryPublication . Rodrigues, Ana Cristina; Martins, Daniela; Duarte, Maria Salomé; Marques, Susana; Gama, Miguel; Dourado, Fernando; Carvalho, Ricardo; Cavaleiro, AnaABSTRACT: The production cost of bacterial nanocellulose (BNC) is a major limitation to its widespread use. However, this limitation can be addressed by using alternative low-cost substrates and high-yield strains. Agro-industrial wastederived substrates offer a cost-effective and sustainable solution, but their high organic load often requires additional downstream wastewater treatments. Here, we optimized static BNC production using eucalyptus bark hydrolysate (EBH) as a low-cost carbon source and proposed a circular approach for wastewater management. Optimization was performed using response surface methodology - central composite design. The optimized EBH medium yielded a 39.7-fold increase compared to standard medium, with a maximum BNC production of 8.29 f 0.21 g/L. Fermentation wastewater only (WaF) and combined with BNC washing streams (WaW) revealed high levels of organic matter, namely chemical oxygen demand (COD) of 159.0 f 2.0 and 41.1 f 0.3 g/L, and volatile solids (VS) of 99.5 f 0.9 and 26.3 f 0.2 g/L, respectively, requiring treatment before disposal. A sequential anaerobic-aerobic digestion was investigated for wastewater treatment and valorisation. Anaerobic digestion proved to be effective in treating the wastewater: methanization percentages over 87 % were achieved, and methane productions of 486 f 2 and 544 f 30 L/kg VS were obtained from WaF and WaW, respectively. Subsequent aerobic treatment was unsuccessful in further reducing COD levels (approximately 1.5 g/L). Notably, treated wastewater was recycled into the production process up to 45 % without affecting the BNC yield. This study provides valuable insights into the optimization of BNC production from lignocellulosic biomass and the management of wastewater streams, contributing to the development of a more sustainable and economically viable process.