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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.
- Biopolymers Derived from Forest Biomass for the Sustainable Textile IndustryPublication . Dias, J. C.; Marques, Susana; Branco, Pedro C.; Rodrigues, Thomas; Torres, Cristiana A.V.; Freitas, Filomena; Evtuguin, Dmitry; Silva, CarlaABSTRACT: In line with environmental awareness movements and social concerns, the textile industry is prioritizing sustainability in its strategic planning, product decisions, and brand initiatives. The use of non-biodegradable materials, obtained from non-renewable sources, contributes heavily to environmental pollution throughout the textile production chain. As sustainable alternatives, considerable efforts are being made to incorporate biodegradable biopolymers derived from residual biomass, with reasonable production costs, to replace or reduce the use of synthetic petrochemical-based polymers. However, the commercial deployment of these biopolymers is dependent on high biomass availability and a cost-effective supply. Residual forest biomass, with lignocellulosic composition and seasonably available at low cost, constitutes an attractive renewable resource that might be used as raw material. Thus, this review aims at carrying out a comprehensive analysis of the existing literature on the use of residual forest biomass as a source of new biomaterials for the textile industry, identifying current gaps or problems. Three specific biopolymers are considered: lignin that is recovered from forest biomass, and the bacterial biopolymers poly(hydroxyalkanoates) (PHAs) and bacterial cellulose (BC), which can be produced from sugar-rich hydrolysates derived from the polysaccharide fractions of forest biomass. Lignin, PHA, and BC can find use in textile applications, for example, to develop fibers or technical textiles, thus replacing the currently used synthetic materials. This approach will considerably contribute to improving the sustainability of the textile industry by reducing the amount of non-biodegradable materials upon disposal of textiles, reducing their environmental impact. Moreover, the integration of residual forest biomass as renewable raw material to produce advanced biomaterials for the textile industry is consistent with the principles of the circular economy and the bioeconomy and offers potential for the development of innovative materials for this industry.
- Production of sustainable aviation fuel precursors using the oleaginous yeast Rhodotorula toruloides PYCC 5615 cultivated on eucalyptus bark hydrolysatePublication . Saraiva Lopes da Silva, Maria Teresa; Dutra, Francisca; Gomes, Miguel; Costa, Paula; Paradela, Filipe; Ferreira, Frederico Castelo; Torres Faria, Nuno Ricardo; Mugica, Paula; Pinheiro, Helena M.; Sá-Correia, Isabel; Gírio, Francisco; Marques, SusanaABSTRACT: Sustainable aviation fuels (SAF) obtained from renewable sources of carbon can reduce carbon dioxide emissions and contribute for mitigating climate changes. In the present study, the yeast Rhodotorula toruloides PYCC 5615 was found to be highly promising for the bioconversion of eucalyptus bark hydrolysate and the accumulation of intracellular lipids which were further thermochemically processed to bioenergy intermediaries for SAF production. Two growth medium formulations were tested. Eucalyptus bark hydrolysate, obtained by steam explosion followed by enzymatic hydrolysis, was supplemented with yeast nitrogen base medium or with corn steep liquor and mineral medium. The latter produced the highest fatty acid content and productivity (30 % w/w and 0.11 g/ (L.h) respectively). Thereafter, the whole yeast biomass (WB) and the de-oiled biomass (DOB), obtained after lipid extraction, were processed into Bio-crude using a hydrothermal liquefaction (HTL) reactor, with a yield of approximate to 40 % (w/w). The two obtained Bio-crude fractions and the yeast lipids fraction (YL) were further upgraded by hydrodeoxygenation (HDO), to remove oxygen atoms and increase the hydrocarbon content, resulting in a Bio-crude composed of linear long-chain fatty acids suitable for processing to SAF. The best Bio-crude characteristics was observed for WB and YL fractions, with 34.8 % and 40.7 % of hydrocarbons, respectively. Both WB and YL hydrocarbons were composed of C15-C17 compounds. These results demonstrate the potential of an integrated process based on microbial oils from R. toruloides PYCC 5615 to produce SAF precursors from Eucalyptus bark residues, contributing for the sustainable jetfuel bioproduction process.
- Alternative feedstocks for high-quality biodiesel: Lipid production from eucalyptus bark hydrolysate by Yarrowia lipolytica W29 using different cultivation modesPublication . Dias, Bruna; Lopes, Marlene; Marques, Susana; Gírio, Francisco; Belo, IsabelABSTRACT: Microbial lipids produced by yeasts from lignocellulosic biomass are a promising feedstock for the biodiesel industry, providing a renewable energy source as an alternative to traditional fossil fuels. This study investigated the potential of Yarrowia lipolytica W29 to produce lipid-rich biomass from undetoxified sugar-concentrated eucalyptus bark hydrolysate (EBH). The lipid concentrations achieved in batch cultures (13.4 g L-1) were the highest for wild-type Y. lipolytica strains in lignocellulosic hydrolysates. Different two-stage cultivation modes (repeated batch, continuous-feeding fed-batch, and pulse fed-batch) were studied to enhance biomass and lipid production. The cell and lipid mass was higher in pulse fed-batch and continuous-feeding fed-batch cultures than batch cultures. Production of citric acid, a side product of industrial interest, was improved in the continuous-feeding fed-batch culture. Microbial lipids produced by Y. lipolytica W29 were highly unsaturated and mainly composed of oleic acid (50% to 53%). The estimated properties of the biodiesel that would be obtained from these intracellular lipids would meet the international biodiesel standards EN 14214 and ASTM D6751. This study demonstrates the feasibility of using EBH for Y. lipolytica lipid production and promotes the sustainable production of high-quality biodiesel from lignocellulosic feedstocks.
- Sustainable Production of Poly(3-hydroxybutyrate) Using Eucalyptus Bark: Integration with Green Downstream ProcessingPublication . Matias, João; Rodrigues, Thomas; Torres, Cristiana A. V.; Marques, Susana; Ribeiro, Belina; Gírio, Francisco; Reis, Maria A.; Freitas, FilomenaABSTRACT: This study integrates the valorization of a lignocellulose material into poly(3-hydroxybutyrate), P(3HB), with biopolymer extraction from bacterial cells with the enzyme alcalase. The work focused on Burkholderia thailandensis DSM 13276 as the P(3HB) producer and on eucalyptus bark, a byproduct from the pulp industry, as the sole feedstock for bacterial cultivation. The eucalyptus bark was hydrolyzed by a cellulolytic enzymatic cocktail following steam explosion and further subjected to ultrafiltration for enzyme recovery. The resulting hydrolysate supported good cell growth, achieving a cell dry weight of 7.67 +/- 0.16 g/L within 72 h of cultivation, and high P(3HB) content (60.0 +/- 2.19 wt %) in the bacterial cells, clearly favoring biopolymer synthesis over cell growth, as demonstrated by the polymer and growth yields (0.190 gP(3HB)/gsugar and 0.026 gX/gsugar, respectively). High extraction efficiency (96%) and biopolymer purity (100 +/- 3.38%) were reached by enzymatic treatment, resulting in a sample with properties aligned with those of commercial P(3HB) in terms of molecular mass distribution, crystallinity, and thermal properties. These findings demonstrate the successful use of a sustainable feedstock together with the application of environmentally friendly technologies based on the use of enzymes for both lignocellulosic saccharification and biopolymer recovery to develop high-quality bioplastics, advancing the goals of a circular bioeconomy.
- Nanobubble-enhanced oxygen transfer in bacterial nanocellulose production: Comparative evaluation with static and airlift systemsPublication . Rodrigues, Ana Cristina; Martins, Daniela; Carvalho, Ricardo; Marques, Susana; Belo, Isabel; Espina, Begona; Dourado, Fernando; Gama, MiguelABSTRACT: Despite the unique properties of bacterial nanocellulose (BNC), oxygen limitation during large-scale production impairs microbial metabolism and cellulose synthesis, leading to high production costs and limited commercial success. Static fermentation can achieve high titers, but industrially it is operationally challenging. Agitated systems like airlift (AL) bioreactors, allow faster production but typically yield lower titers. This study pioneered the use of an agitated bioreactor equipped with a nanobubble (NB) generator, and its performance was compared with that of static and AL systems, employing a newly isolated Komagataeibacter sp. strain from kombucha cultivated in Eucalyptus bark hydrolysate and corn steep liquor. Key monitored parameters included dissolved oxygen, cell density, pH, sugar and lactic acid contents, and BNC production. The obtained BNC was characterized for its crystallinity, thermal stability, degree of polymerization, morphology and fiber size. The AL-and NB-derived BNC exhibited a denser network structure, lower crystallinity index, and lower polymerization degrees than that from static culture. NB technology generated stable nanobubbles (size: 95.8 f 12.9 nm; zeta potential:-14.2 f 8.6 mV). At 1 L. min-1 airflow, compared to AL, the NB bioreactor achieved a 6-fold higher volumetric mass transfer coefficient (kLa 35.9 f 1.2 h-1) and oxygen transfer rate (OTR: 309.7 f 10.2 mg.L-1.h-1). It supported greater cell density but maintained a similar BNC volumetric productivity to that of the AL (0.023 g.L-1.h-1), and moderately higher (near 280%) than that of static culture (0.0082 g.L-1.h-1). Thus, the improved oxygen levels provided by the NB system favored biomass growth rather than BNC production, suggesting that further optimization is needed to redirect carbon flux toward BNC production.