| Congresso Brasileiro de Microbiologia 2023 | Resumo: 101-1 | ||||
Resumo:In Brazil, the cultivation of sugarcane for sugar and alcohol production results in the largest quantity of agricultural waste. However, utilizing these lignocellulosic residues for biotechnology purposes presents a challenge due to their complex structures, which require specific treatments to break down the lignin responsible for the resistance to chemical and biological degradation of bagasse fiber. Among the various chemical oxidizing agents, ozone is highly reactive on substances with functional groups containing high electron density, such as the alkene bonds present in lignin. It has numerous advantages, including the ability to convert lignin into derivative compounds, produce no toxic residues, and conduct reactions at ambient temperature and pressure.
Washed, dried, crushed, and sieved sugarcane bagasse samples weighing 20g (ranging between 1.41 and 2.83mm) underwent ozonolysis at a flow rate of 0.624 mg/min-1 for 10 minutes using a rotation scheme, ensuring equal exposure to ozone for all biomass during the designated time period. The pre-treated samples were then submitted to hydrothermal hydrolysis, where ultrapure water was used in a proportion of 95mL for each 5g of ozonated bagasse. The experiment was conducted in 250mL Erlenmeyer flasks, closed with aluminium foil, in an autoclave for one hour at 1 atm. At the end, the samples were filtered, with the liquid stored and the solid discarded.
To test the capacity of Micrococcus luteus to convert lignin-derived compounds (primarily ferulic acid) into vanillin and related substances (such as acetovanillone), we employed the hydrolysates obtained from the previous step and used them in fermentations. In each trial, we added 10 or 50% of bagasse hydrolysates to 15mL of LB culture medium, along with a 10μL bacterial inoculum (OD 600 nm = 0.8). The mixture was incubated at 35°C and 150 rpm for ten hours, and every two hours, 200μL aliquots were collected from the fermentation medium for absorbance-based growth determination and 2mL aliquots were removed to prepare the samples for chromatographic analysis and pH measurement. The aliquots collected during fermentation and the initial hydrolysates were analysed through high performance liquid chromatography (HPLC), using 5 mM ammonium formate + 0.1% formic acid in water as solvent “A” and Solvent “B” 5 mM ammonium format + 0.1% formic acid in methanol.
We verified the efficacy of the treatment and its ability to yield derivative compounds that can be metabolized by bacteria to produce vanillin. Micrococcus luteus, in particular, produced 145 mg/L of vanillin after 10 hours of cultivation using hydrothermally treated compounds, suggesting the promising potential of microbial culture in obtaining economically important compounds like vanillin and acetovanillone from hydrolysed sugarcane bagasse (Figure 1). Furthermore, ozone pre-treatment itself generated substances like coumaric acid (32 mg/L) and acetovanillone (21 mg/L), which can serve as end-products or precursors for other compounds in the industry (Figure 2).
Figure 1. Overview of compound concentration for M. luteus cultures with liqueurs hydrothermally extracted from bagasse subjected to ozonolysis for 10 minutes. Cultures had 10% or 50% liquor concentration. Values expressed in mg/L.
Figure 2. Quantification of compounds in sugarcane bagasse extracts treated with ozonolysis for 10 and extracted with water. Values expressed in mg/L.
Palavras-chave: Bioconversion, vanillin, ozonolysis, ferulic acid, acetovanillone Agência de fomento:CAPES - Coordenação de Aperfeiçoamento de Pessoas de Nível Superior | |||||