Prospecção de bactérias produtoras de compostos ativos de superfície e enzimas degradativas de hidrocarbonetos, com potencial de utilização em processos de biorremediação

Abstract

Nowadays, there is a great concern regarding the waterbodies contamination by industrial wastewaters, especially the oily ones. The biological treatment of these wastewaters and other oil contaminated areas is increasingly being distinguished mainly because of its simplicity, low costs and lower environmental impacts. Furthermore, these wastewaters can present a large microbial diversity exhibiting interesting adaptations for applications in industrial processes, which reinforces the importance of microbial prospecting of this material. Microorganisms with the potential for oil hydrocarbons degradation and/or for production of Surface Active Compounds (SAC) and hydrolitic/oxidative enzymes were isolated from oily wastewaters from a facultative lagoon located on a industrial wastewaters treatment plant. The oily wastewaters were characterized regarding their total bacterial population and the hydrophobic compounds degrading ones (motor oil, paraffin and kerosene) and regarding the metabolic diversity against 31 carbon sources using the Biolog-ECOPLATE system. The total heterotrophic population was 3,36X107 NMP mL-1 and the degrading bacteria one for motor oil, paraffin and kerosone corresponded to 0,49%, 0,017% e 0,036% of this total, respectively. The metabolic diversity in the community was high, consuming 30 carbon sources expressively in the first 48 hours. A total of 112 bacteria were isolated with and without using the enrichment technique in mineral broth supplemented with hydrocarbons. 82 of these isolates were identified sequencing regions of the 16S rRNA gene. The prevailing observed genus was Bacillus sp., being also found Rhodococcus sp., Enterobacter sp., Staphylococcus sp., Leucobacter sp., Stenotrophomonas sp., Ochrobactrum sp., Acinetobacter sp., Chryseobacterium sp., Klebsiella sp., Escherichia sp. Pseudomonas sp., Shewanella sp., Alcaligenes sp., Enterococcus sp. and Kerstersia sp. and the relative frequency of them varied according to the selection method. The isolates were evaluated regarding their capacity to grow and produce SAC in mineral broth containing crude oil and glucose. The glucose essays obtained the highest growth means (expressed in optical density at 600 nm and dried biomass in mg mL-1), but nearly 10% of the isolates was also able to perform a high growth in the medium supplemented with oil (O.D. 600 nm > 1.5). The source which provided the highest means of surfactant/emulsifying activity was glucose and 24% of the isolates presented emulsifying activity only using this source, 12% only using oil and 64% using both sources. The emulsifying index varied from 0 to 76%, being the highest indexes, above 60%, obtained from 15 isolates using glucose (13,4%) and 12 (10,7%) using oil. The surfactant activity of the isolates, evaluated by the drop spreading, varied from 0 to 2.2 cm and 3 isolates (2,7%) promoted the best results, with a spreading above 1.8 cm. The surfactant activity, evaluated by the surface tension decay, varied from 68 mN m-1 to 29.9 mN m-1, and 4 of the isolates (3,9%) promoted the best results, reducing the tension to values below 35,0 mN m-1. In the evaluation of 70 isolates regarding the potential to degrade hydrocarbons, it was possible to confirm the catechol 1,2-dioxigenase enzyme activity in 8 isolates and of groups II and III alcane hydroxylases in 10 and 14 isolates, respectively. The activity of esterases, lipases, terminal epoxide hydrolases, non-terminal epoxide hydrolases and groups 1 and 3 Baeyer-Vileger monooxygenases were detected in 5, 16, 6, 7, 5 and 12 isolates, respectively. The isolates Bacillus subtilis LAPER 90 and LAPER 93 and Alcaligenes sp. LAPER 94 produced respectively 1.85 g L-1, 0.95 g L-1 e 1.1 g L-1 of the SAC when grown in mineral broth supplemented with 2% (g/v) of glucose. The crude extracts, obtained through the precipitation with ethanol, formed stable emulsions with aliphatic hydrocarbons (hexane and hexadecane), aromatics (toluene and xylene) and oil products (kerosene, motor oil and Diesel fuel). These biomolecules presented in their composition variable percentages of proteins (3.08 to 38.52%), carbohydrates (8.79 to 50.40%) and lipids (16 to 40%). Another feature evaluated was the capacity to degrade hydrocarbons and remove crude oil during the cultivation in mineral broth supplemented with this substratum. From 10 evaluated isolates, 6 presented oil removal rates between 37 and 90%, being the highest one observed for Acinetobacter junni LAPM 30.