| Congresso Brasileiro de Microbiologia 2023 | Resumo: 210-1 | ||||
Resumo:Biodiesel is a renewable biofuel with a lower greenhouse gases emission balance compared to diesel, making it a less polluting fuel in the economy. However, biodiesel production generates a contaminated residue called generically as crude glycerol (CG), but it contains also methanol, fatty acids, soup, and minerals. Glycerol can serve as a carbon source for microbial growth and has been used as substrate in several bioprocesses. However, the presence of other contaminants in CG can hinder its use in bioprocesses. This work aims to adapt a Clostridium beijerinckii strain to the biodiesel residue as substrate to produce of a high-value product, 1,3-propanediol (1,3-PDO), a monomer of industrial polymer.
The high toxicity of CG renders it incapable to being consumed by the majority of the microorganisms, necessitating the use of multiple strategies to enable microbial growth in media containing crude glycerol.
Adaptative Laboratory Evolution (ALE) is a methodology used to enhance the growth capacity of microorganism in medium containing stressful compounds by acquiring resistance or the ability to assimilate the compound. The methodology involves introducing the microorganism into a low concentration of toxic compound medium and replacing the inoculum multiple times to accumulate adaptation in the microorganism. After the initial adaptation, the concentration of the toxic compound is increased, and the process continue.
The objective of this work is to conduct an ALE of Clostridium beijerinckii Br21, a bacterium capable of utilizing glycerol and producing 1,3-PDO, but which demonstrates poor growth in CG media.
Here, 5, 10, and 20% (v/v) of CG was added to a culture medium named WISmod. The bacterial growth was determined by optical density (OD) at 600 nm, and the glycerol and 1,3-PDO concentrations were quantified by gas chromatography.
Initially, in the 5% CG medium, C. beijerinckii Br21 took 48 hours to growth. After 8 cycles of re-inoculum, the period needed to the bacteria growth was reduced to 24 hours and OD increased to 0.236 and 0.3 g L-1 glycerol was consumed.
At this point, the inoculum was introduced into a 10% CG medium. After 12 cycles of 24 hours inoculum and growth, OD was increased to 0.873, and 0.8 g L-1 of glycerol was consumed with 0.247 g L-1 of 1,3-PDO.
Finally, the previous adapted bacterial culture was introduced into a 20% CG medium. The growth was retarded by 48 hours. However, after 4 inoculum cycles, the period needed to growth was reduced to 24 hours. At the 8th cycle, the OD increased to 1.68 with 2.9 g L-1 glycerol consumption, i.e., 7.1 and 9.6 times higher compared to the non-adapted microorganism. The final concentration of 1,3-PDO was 0.34 g L-1, representing a 37% increase comparted to the last cycle in 10% CG.
Further, the genomic DNA was isolated and sent to sequencing to compare the DNA sequences of the adapted and the original strain. The methodology for the C. beijerinckii Br21 adaptation to CG enhances its growth capacity in crude glycerol media, reinforcing its resilience against toxic compounds.
Palavras-chave: Adaptative Laboratory Evolution, biodiesel, Crude Glycerol, Clostridium beijerinckii, 1,3-propanediol Agência de fomento:Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), grant number 88887.701729/2022-00. Fundação de amparo a pesquisa do Estado de São Paulo (FAPESP), grant number 2022/040240-0. | |||||