Butanol Production by Clostridium beijerinckii using green cococut fiber lignocellulosic residue
lignocellulosic material, cellulase recycling, pre-treatment, bacteria, ABE fermentation, biobutanol.
The search for renewable energies that minimize the environmental damage caused mainly by the burning of fossil fuels has supported studies for the diversification of the global energy matrix. In the case of Brazil, the insertion of new biofuels such as butanol associated with the valorization of lignocellulosic biomass presents an opportunity to consolidate its historical vocation in the leadership of renewable energy sources, especially when the world seeks solutions to mitigate climate change. The green coconut shell, for example, is an abundant agro-industrial and urban waste that can be transformed into the substrate and then converted into products of industrial value, adding value to the production chain, and solving environmental problems related to its disposal. In this context, we investigated the potential of the green coconut husk in the production of cellulosic butanol (or biobutanol) by ABE (Acetone-Butanol-Ethanol) fermentation, using Clostridium beijerinckii. Initially, enzymatic hydrolysis tests were carried out to extract information about the release of sugars from green coconut shells (GCS) and the ability to recycle commercial cellulolytic cocktails. The behavior and recovery of cellulolytic enzymes remaining from GCS delignified by different pre-treatments (dilute acid, alkaline, and acid-alkaline) were evaluated. At another time, batch fermentation tests investigated the influence of some nutrients (nitrogen sources and mineral medium) on hydrolyzed supplementation to produce butanol and other solvents. Adsorption and desorption studies involving two types of cellulases (Trichoderma reesei ATCC 26921 and Cellic Ctec2, 10 FPU/g, initial dosage) with 5% solids loading showed that CCV delignified by alkaline (LCM1) and acid-alkali pretreatments (LCM3) showed lower cellulase adsorption capacity (48% and 69%). The LCM3 pretreatment provided the highest recovery of cellulase by desorption without the addition of Tween 80 (reaching 50%). Cellulases bound to the solid residue and dissolved in the supernatant after enzymatic hydrolysis (HE) can be reused in a new cycle without compromising the sugar yield results. Using GCS as a substrate, the addition of the solid residue and the supernatant from the previous cellulase adsorption stage reached satisfactory sugar yield (73% for LCM1 and 60% for LCM3) only using the recycled enzyme (without the addition of enzyme) concerning the control assay. Thus, it was possible to recycle the cellulases in two successive cycles of HE with reduced enzymatic load to reach the same yields of reducing sugars (up to 84%), allowing cheaper and more efficient hydrolysis of lignocellulosic biomass. ABE fermentation assays evaluated the influence of some nutrients, nitrogen sources, and minerals on hydrolyzed supplementation to produce butanol and other solvents. It was found that ~9.0 g/L of the initial substrate (glucose + xylose) present in the hydrolyzate resulted in an ABE yield of 0.53 g/g of sugars1, with 3.4 g/L of butanol produced in 96 h of fermentation, using the initial inoculum concentration of 1.0 g/L. The absence or insufficiency of some nutrients (minerals and phosphate buffer) resulted in low ABE production, indicating the relevance of the adequacy of supplements to the chosen fermentation medium and the type of microorganism used. Fed-batch fermentation resulted in higher butanol and ABE yields (0.08 g/L.h and 0.1 g/L.h, respectively), despite lower butanol and ABE yields (0.21 g/g and 0.24 g/g, respectively). The results indicate that green coconut waste has energy potential and is the low-cost raw materials for the biotechnological production of butanol, as an alternative and renewable product.