Biodegradação de gasolina e óleo diesel utilizando biossurfactantes e Pseudomonas putida com plasmídio TOL
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Data
2017-09-06
Autores
Claro, Elis Marina Turini [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Grande atenção vem sendo dada a contaminação por hidrocarbonetos resultante das atividades da indústria petroquímica. Dentre as tecnologias utilizadas na remediação e tratamento de áreas impactadas, a biodegradação vem despontando como um dos principais campos de pesquisa em microbiologia, se apresentando como uma técnica bastante eficaz. Dessa forma, o presente trabalho visou estudar a biodegradação de gasolina, óleo diesel e dos compostos benzeno, tolueno, etilbenzeno e o-xileno (BTE(o-)X) por Pseudomonas putida CCMI 852 com plasmídeo TOL pWW0, com aplicação de biossurfactante, para aumentar a biodisponibilidade de compostos hidrofóbicos, produzido por Pseudomonas aeruginosa LBI a partir do soro de ricota, como fonte de carbono e com avaliação da toxicidade com sementes de alface (Lactuca sativa) e genotoxicidade com sementes de cebola (Allium cepa), antes e após a biodegradação. Foi observado um potencial de produção de biossurfactantes do tipo ramnolipídios em soro de ricota como substrato alternativo para produção, com rendimento de 0,45 g L-1 . O monitoramento da biodegradação dos hidrocarbonetos, foi realizado por ensaios colorimétricos utilizando os indicadores redox DCPIP e TTC e a quantificação foi realizada por cromatografia gasosa acoplada com espectrometria de massa (CG/MS). Os testes colorimétricos foram eficientes e bastante eficaz para verificar a atividade bacteriana, indicando o potencial do microorganismo em degradar esses compostos, fato este que comprovou-se pela análise em GC/MS. O plasmídeo TOL pWW0 encontrado naturalmente na linhagem bacteriana P. putida CCMI 852, confere às células a capacidade de degradarem hidrocarbonetos presentes na gasolina mais facilmente em relação ao óleo diesel e que a P. putida CCMI 852 não foi capaz de metabolizar o benzeno, nem individualmente, nem em misturas, indicando a via de degradação da linhagem sendo a via TOL. Em mistura BTE(o-)X, a presença do benzeno ocasionou efeito negativo na degradação do tolueno que apresentou taxa de degradação 33,5% menor do que quando individualmente, enquanto que em mistura, o etilbenzeno aumentou 50,3%. Em misturas ternárias TE(o-)X, os compostos apresentaram maior taxa de degradação em relação à presença do benzeno, com aumento de 14,2% para tolueno, seguido de 4,8% e 2,5% para o (o-)xileno e etilbenzeno, respectivamente. A sequência de degradação encontrada foi tolueno, o-xileno e etilbenzeno. O uso do biossurfactante no processo, não aumentou a taxa de biodegradação. Contudo, ao realizar testes de toxicidade (com sementes de alface) foi observado que na maioria dos ensaios foram produzidos compostos intermediários da biodegradação, mais tóxicos aos organismos-teste do que a substância original. Os testes com A. cepa permitiram verificar potencial genotóxico da gasolina e do óleo diesel biodegradados após 10 dias, por alterações no índice mitótico e mutagênico dos compostos.
Great attention has been given to the contamination by hydrocarbons from the petrochemical industry. Among the technologies used in the remediation and treatment of impacted areas, biodegradation has emerged as a very effective technique, which is now among the main research fields in microbiology. Thus, this thesis aimed to study the biodegradation of benzene, toluene, ethylbenzene and o-xylene (BTE(o-)X) by Pseudomonas putida CCMI 852 with the TOL pWW0 plasmid, as well as biosurfactant application to increase the bioavailability of hydrophobic compounds produced by Pseudomonas aeruginosa LBI using ricotta serum as the source of carbon. The evaluation of toxicity with lettuce seeds (Lactuca sativa) and genotoxicity with onion seeds (Allium cepa), before and after biodegradation, was also considered. The potential production of rhamnolipidic biosurfactants in ricotta whey as an alternative substrate yielded 0.45 g L-1. Monitoring the biodegradation of hydrocarbons was performed by colorimetric assays using the both DCPIP and TTC redox indicators. Quantification was performed by gas chromatography coupled with mass spectrometry (GC / MS). The colorimetric tests were efficient and quite effective to verify the bacterial activity, indicating the potential of the microorganism to degrade these compounds. This fact was evidenced by GC / MS analysis. The plasmid TOL pWW0 found naturally in the bacterial line P. putida CCMI 852, gives the cells the ability to degrade hydrocarbons present in gasoline more easily relative to diesel oil. P. putida CCMI 852 was not able to metabolize benzene, nor individually, or in mixtures, indicating that the route of degradation of this specific strain is the TOL route. In the BTE(o-)X mixture, benzene caused a negative effect on the degradation of toluene, which had a degradation rate of 33.5% lower than its individual presence. While in the mixture, ethylbenzene increased its degration rate by 50.3%. In ternary mixtures TE(o-)X, the compounds had a higher rate of degradation in relation to the presence of benzene, with a 14.2% increase in toluene, followed by 4.8% and 2.5% for xylene and ethylbenzene, respectively. The degradation sequence found was toluene, o-xylene and ethylbenzene. The use of the biosurfactant in the process did not increase the rate of biodegradation. However, when performing toxicity tests (with lettuce seeds) it was observed that in the majority of the assays, intermediate compounds of the biodegradation were produced, which were more toxic to the test organisms in the original substance. The tests with oniun seeds verified the genotoxic potential of biodegraded gasoline and diesel oil after 10 days, due to changes in the mitotic and mutagenic index of such compounds.
Great attention has been given to the contamination by hydrocarbons from the petrochemical industry. Among the technologies used in the remediation and treatment of impacted areas, biodegradation has emerged as a very effective technique, which is now among the main research fields in microbiology. Thus, this thesis aimed to study the biodegradation of benzene, toluene, ethylbenzene and o-xylene (BTE(o-)X) by Pseudomonas putida CCMI 852 with the TOL pWW0 plasmid, as well as biosurfactant application to increase the bioavailability of hydrophobic compounds produced by Pseudomonas aeruginosa LBI using ricotta serum as the source of carbon. The evaluation of toxicity with lettuce seeds (Lactuca sativa) and genotoxicity with onion seeds (Allium cepa), before and after biodegradation, was also considered. The potential production of rhamnolipidic biosurfactants in ricotta whey as an alternative substrate yielded 0.45 g L-1. Monitoring the biodegradation of hydrocarbons was performed by colorimetric assays using the both DCPIP and TTC redox indicators. Quantification was performed by gas chromatography coupled with mass spectrometry (GC / MS). The colorimetric tests were efficient and quite effective to verify the bacterial activity, indicating the potential of the microorganism to degrade these compounds. This fact was evidenced by GC / MS analysis. The plasmid TOL pWW0 found naturally in the bacterial line P. putida CCMI 852, gives the cells the ability to degrade hydrocarbons present in gasoline more easily relative to diesel oil. P. putida CCMI 852 was not able to metabolize benzene, nor individually, or in mixtures, indicating that the route of degradation of this specific strain is the TOL route. In the BTE(o-)X mixture, benzene caused a negative effect on the degradation of toluene, which had a degradation rate of 33.5% lower than its individual presence. While in the mixture, ethylbenzene increased its degration rate by 50.3%. In ternary mixtures TE(o-)X, the compounds had a higher rate of degradation in relation to the presence of benzene, with a 14.2% increase in toluene, followed by 4.8% and 2.5% for xylene and ethylbenzene, respectively. The degradation sequence found was toluene, o-xylene and ethylbenzene. The use of the biosurfactant in the process did not increase the rate of biodegradation. However, when performing toxicity tests (with lettuce seeds) it was observed that in the majority of the assays, intermediate compounds of the biodegradation were produced, which were more toxic to the test organisms in the original substance. The tests with oniun seeds verified the genotoxic potential of biodegraded gasoline and diesel oil after 10 days, due to changes in the mitotic and mutagenic index of such compounds.
Descrição
Palavras-chave
Biodegradação, Hidrocarbonetos, BTEX, Biossurfactante, Ecotoxicidade, Biodegradation, Hydrocarbons, Biosurfactant, Ecotoxicity