Desenvolvimento de biomoléculas para a biorremediação de solos contaminados em Portugal

The main objective of this work is to implement an innovative, sustainable and environmentally friendly alternative for the bioremediation of soil and water in Portugal, mainly contaminated with hydrocarbons and derivatives, contributing to environmental sustainability. To achieve this great objective, several specific and mensurable objectives were delineated: I. Characterize the contaminated soil and water through analytical techniques. This stpe is very important to better understand which contaminants are present in the samples. Therefore, analytical methods will be used to qualify and quantify the contaminants. All the analytical techniques that cannot be in-house preformed will be subcontracted in certified laboratories. II. Develop a formulation to correct the micronutrients content and pH value of the contaminated sites to promote microbial growth. Taking in consideration the characterization of the samples, a formulation should be produced to enhance the bioremediation of the soil and allow the microorganisms to thrive. This will be a natural solution, and agrifood waste will be exploited. III. Evaluate the efficiency of several biosurfactants already in the market for bioremediation purpose. Several biosurfactants are already available in the market. In this task, the capacity of different biosurfactants to emulsify hydrocarbons will be evaluated, namely by the surface tension between the water phase and a hydrocarbon phase in the presence of different biosurfactants. IV. Evaluate the impact of biosurfactants in the microbial ecosystem of contaminated sites. The addition of biosurfactant in different concentrations to contaminated soil/water may impact the microbial ecosystem existent in these samples. This impact can be measured by CFUs count and compared to the addition of biosurfactants. Moreover, the diversity of species presents before and after the addition of biosurfactant will be evaluated through 16S rRNA gene and identification of the microorganisms present in those samples through a metagenomics approach. V. Low-medium scale in-house production of the most suitable biosurfactant for bioremediation taking into consideration the previous points. For this task, two approaches will be followed: i) the production of biosurfactant using strains already described with this capacity; ii) the production of biosurfactant in a different host using heterologous expression. Heterologous expression allows the production of biosurfactants in different hosts most suitable for production. In this work, the genes coding to produce the most suitable biosurfactant type will be inserted through a vector into Pseudomonas putida and E. coli (heterologous expression) allowing the production of the biosurfactant with a higher yield than the wild-type strains, theoretically. For P.putida, the vectors pSEVA, designed specifically for this strain will be used. For E. coli, expression vectors based on the pET expression vectors will be used. Both types of vectors are already available in-house, as well as the hosts. Moreover, the hypothesis of using yeast for heterologous expression may be considered, depending on the type of biosurfactant that will be produced. In this case, S. cerevisiae may be used together with, for instance, pYES expression vectors. Moreover, for better stability and production yields an approach of gene integration in the genome of any host may be considered and performed using the Crispr-Cas9 system.