|591-1||Selection of polyhydroxyalkanoates producing bacteria from sugarcane rhizosphere soil by the colony PCR and Sudan Black staining|
|Autores:||Cláudia Elizabete Pereira Lima (UFPE - Universidade Federal de Pernambuco) ; Juliana de Castro Nunes Pereira (UFPE - Universidade Federal de Pernambuco) ; Paula Débora de Melo Brasileiro (UFPE - Universidade Federal de Pernambuco) ; Romero Marinho Moura (UFPE - Universidade Federal de Pernambuco) ; Idjane Santana Oliveira (UFPE - Universidade Federal de Pernambuco) |
The rhizosphere has a large microbial diversity, with excellent polyhydroxyalkanoates (PHAs) producing bacteria, biodegradable polymers used in the manufacture of biodegradable plastics. This work aimed to isolate PHA producing bacteria from sugarcane rhizosphere soil, in northeastern Brazil, using colony PCR and Sudan Black staining. Composite samples consisting of 10 subsamples from sugarcane rhizosphere soil were collected in the city of Carpina, PE. Serial dilutions were made in saline to 10-5. The dilutions were plated on nutrient agar medium (NA) and incubated for 48 hours at 37 °C. The potential to synthesize PHA was tested qualitatively by Sudan Black staining of colonies growing in medium NA and colony PCR (amplification ofphaC gene). The colony DNA was extracted by boiling method using 50 μL of TE and a strap of the colony plate removal. The colony was amplified by PCR using primers to the phaC gene (DG and DR to amplify DNA fragment 551pb) in a PCR reaction mix (25 μL) containing 0.5 mM dNTP mix, 1. 25 mM MgCl2, 0.3 mM of each primer and 0.25 IU of Taq and 2μL of DNA extracted from the colony. The amplified DNAs were separated in gel electrophoresis (1.0%), stained with Syber safe and visualized with blue light transilluminator. Were used as positive control of PCR, DNA of the bacteria already used in the laboratory to production of PHAS,Cupriavidus necator, Pseudomonas putida and Bacillus thuringiensis israelensis (Bti) and negative control the PCR mix without DNA. Of the 47 bacterial colonies isolated, five (10.6%) showed strong Sudan Black staining and were positive for PCR of phaC gene. Among the bacterial colonies were found PCR positive in one coccus Gram-negative bacteria, three Gram positive bacilli and one Gram-negative bacilli. The results of this study suggest that the sugarcane rhizosphere soil can be considered as suitable habitat for the occurrence of polyhydroxyalkanoates producing bacteria. The colony PCR and Sudan Black staining tests provided an efficient and rapid detection of these bacteria.
Palavras-chave: bacteria, PCR, PHA, sugarcane