Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms

Abstract

The aim of this study was to analyse the effect of oil contamination and biostimulation (soil pH raise, and nitrogen, phosphate and sulphur addition) on the diversity of a bacterial community of an acidic Cambisol under Atlantic Forest. The experiment was based on the enumeration of bacterial populations and hydrocarbon degraders in microcosms through the use of conventional plating techniques and molecular fingerprinting of samples directly from the environment. PCR followed by denaturing gradient gel electrophoresis (DGGE) was used to generate microbial community fingerprints employing 16S rRNA gene as molecular marker. Biostimulation led to increases of soil pH (to 7.0) and of the levels of phosphorus and K, Ca, and Mg. Oil contamination caused an increase in soil organic carbon (170-190% higher than control soil). Total bacterial counts were stable throughout the experiment, while MPN counts of hydrocarbon degraders showed an increase in the biostimulated and oil-contaminated soil samples. Molecular fingerprinting performed with 16S rRNA gene PCR and DGGE analysis revealed stable patterns along the 360 days of experiment, showing little change in oil-contaminated microcosms after 90 days. The DGGE patterns of the biostimulated samples showed severe changes due to decreases in the number of bands as compared to the control samples as from 15 days after addition of nutrients to the soil. Results obtained in the present study indicate that the addition of inorganic compounds to soil in conjunction with oil contamination has a greater impact on the bacterial community than oil contamination only.

New Group-Specific 16S rDNA Primers for Monitoring Foaming Mycolata During Saline Waste-Water Treatment

“…Owing the small size of the analyzed sequences (200-400 bp), the phylogenetic affiliations and sequence similarity values were interpreted with caution (Evans et al 2004). Nevertheless, the results presented here indicate that the primers do preferentially selectively amplify foaming mycolata 16S rDNA facilitating the detection of this important group of bacteria in saline waste-water samples.…”

“…Direct extractions of microbial DNA from pelleted sludge were performed using the FastPrep System and the Fast DNA SPIN Kit for soil (Evans et al 2004). Total DNA was analyzed by electrophoresis in 1% (w/v) agarose in Tris/Borate/EDTA (TBE) buffer and ethidium bromide staining.…”

“…PCR mixtures were prepared as follows: 5 ll Taq buffer 10Â, 7.5 ll 25 mM MgCl 2 , 1 ll each 10 mM dNTPs, 10 pmol of each primer, 0.5 ll bovine serum albumin (5 mg ml )1 ), 0.5 ll 1% formamide (v/v) and 0.05 U Taq polymerase and sterile-filtered milliQ water was added to make 50 ll (Evans et al 2004). Amplification was performed in accordance with the protocol of Heuer et al (1997) and amplification products analyzed by electrophoresis in 1.4% agarose (w/v) gels in TBE buffer with ethidium bromide staining.…”

“…Denaturing gradient gel electrophoresis was carried out at 40 V, 60°C for 16 h in TAE buffer (Boon et al 2002). After electrophoresis, gels were stained with SYBR Green I nucleic acid gel stain for 40 min (Evans et al 2004). The stained gel visualization was carried out under UV transillumination using the STORM system (Pharmacia, Amersham).…”

Bioremediation of Petroleum Hydrocarbons in Soils

“…Nutrient balance (C and N), pH and moisture content of soil were usually affected as a result of contamination by hydrocarbons (Bundy et al 2002 ) . As a result organisms that can withstand and utilize hydrocarbons could only survive and grow affecting whole community structure (Evans et al 2004 ;Bordenave et al 2007 ;Popp et al 2006 ;Baek et al 2007 ) . For example, g -proteobacteria mainly Pseudomonas species were found to be selectively enriched after the contamination by hydrocarbons such as alkanes, and PAHs followed by a -proteobacteria and b -proteobacteria (Popp et al 2006 ) .…”