Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste

Abstract

ABSTRACT. Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oilcontaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 µg DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria -alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac- teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments.

Methodology Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells

“…The most efficient methods for investigating microbial complexity in sedimentary matrix (sludges) are based on direct (in situ) lysis, as this technique introduces less bias in the analysis (Schneegurt et al, 2003;Luna et al, 2006;Maciel et al, 2009). We tested a number of protocols for extracting DNA from an MFC sludge sample.…”

“…Many direct extraction methods combine chemical and enzymatic treatments and physical procedures, although some of these protocols are tedious and laborious, with numerous steps. In addition, the required enzymes are expensive and inefficient for most Gram-positive bacteria (Bollet et al, 1991;Maciel et al, 2009). Any protocol is universally applicable, since the samples are extremely heterogeneous and contain diverse substances.…”

“…Any protocol is universally applicable, since the samples are extremely heterogeneous and contain diverse substances. Evaluation and modification of available protocols are often required for microbial analysis in the biological samples under study, which include soil, water, sediment, sludge, feces, and other types of samples (Maciel et al, 2009;Ning et al, 2009;Ariefdjohan et al, 2010). The procedure must extract DNA from a large number of different bacteria, and the recovered nucleic acid must be suitable for subsequent molecular techniques, such as endonuclease restriction or Taq polymerase amplification.…”

Remediation of Contaminated Sites

“…This technology involves the application of contaminated material that has been excavated onto the soil surface and periodically tilled to mix and aerate the material [35][36]. The contaminants are degraded, transformed and immobilized by means of biotic and abiotic reactions.…”

Microbial Outlook for the Bioremediation of Crude Oil Contaminated Environments

“…Currently, these studies can be conducted using methods based on DNA sequence analysis, especially analysis of the 16S rDNA gene in bacteria and archaea and the 18S gene in fungi. These genes can be amplified by PCR (Polymerase Chain Reaction) and subsequently characterized by electrophoresis using different molecular techniques such as ARDRA (Amplified Ribosomal DNA Restriction Analysis), T-RFLP (Terminal Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA), RISA (Ribosomal Intergenic Space Analysis), DGGE/TGGE (Denaturing Gradient Gel Electrophoresis / Temperature Gradient Gel Electrophoresis, and SSCP (Single-Strand Conformation Polymorphism to obtain microbial community profiles (Ranjard et al, 2000;Kozdrój & Van Elsas, 2001, Konstantinidis & Tiedje, 2007, Maciel et al 2009). …”