Use of phenotypic and molecular tests for the identification of bacteria with the ability to biodegrade hydrocarbons

Abstract

Petroleum is a composite product which, in addition to being very important economically, also stands out for its potential polluter, through contamination of soil and water, being a challenge for environmental sanitation. An alternative for the removal of these compounds is bioremediation, which consists of biotransforming them into less toxic compounds and in some cases without toxicity. Thus, the characterization of microorganisms capable of degrading these toxic compounds is important for the treatment of generated industrial residues. In a previous project carried out in our laboratory, it was possible to isolate and identify 10 microorganisms from composting that showed the ability to grow in the presence of n-hexadecane, a linear hydrocarbon. Isolates were identified as Aquamicrobium sp (MTZ026 and 027), Bacillus shackletonii (MTZ094), Gordonia sp. (MTZ041, 052, 053, 055 and 056) and Gordonia sihwensis (MTZ095 and 096). It is important to note that none of these microorganisms has a pathogenicity description, a desirable feature for the use of an organism as a bioremediator. Thus, this project aims to standardize and compare two phenotypic tests to evaluate the ability of these isolates to degrade hydrocarbons. The first phenotypic method, called Double Layer, consists of pouring the semi-solid medium containing the hydrocarbon under study onto a layer of solid medium and then seeding the microorganisms. The degradation of the compound by the microorganism will be observed by the presence of a transparent halo around the bacterial colony. The second method to be applied, the 2.6-dichlorophenol indophenol test (2,6-DCPIP), will be the use of an oxy-reduction indicator that detects the oxidation of NADH in NAD +, a reaction that is related to Degradation of bacteria. In addition, the genes coding for alkane-1-monooxygenases, cytochrome P450 (subfamily CYP153) and dioxigenases, described as the main responsible for the initial degradation of hydrocarbons, will be verified by PCR using degenerate oligonucleotides already described in the literature. The M. vanbaalenii reference strain PYR1 DSM7251 will be used as a positive control of the tests. The standardization of phenotypic tests will be useful for screening of isolates, in order to identify microorganisms that are candidates for biotechnological application. It should be noted that our research group participates as a collaborator in the FAPESP Thematic Project no. 2011 / 50870-6 entitled "Study of the microbial diversity of the São Paulo State Zoological Park", which has the general objective of collecting, analyzing and prospecting molecular data of three microbiomes: composting, lake and fawns of howler monkeys. Since then, it investigates microorganisms with the capacity to degrade hydrocarbons from the composting process in order to select at least two microorganisms with characteristics of interest for sequencing their genomes (draft level) and after analyzing the results using bioinformatics techniques, verify the existence of possible products with biotechnological applications. The motivation to analyze the capacity of degradation of hydrocarbons from microorganisms derived from composting was due to the particularity of the Organic Compound Production Unit (UPCO) of the Zoological Park of São Paulo Foundation (FPZSP), which processes organic matter from various origins as: Excrement of approximately 3,500 animals, carcasses, lake water sediments, animal litter remnants, food waste, pruning residues from park gardens and plant remains. Considering these data and knowing that the composting process occurs predominantly by the action of resident microorganisms, it is expected that this ecological niche has microorganisms with the capacity to degrade the most diverse compounds, including hydrocarbons. (AU)