Polyphasic analysis of the bacterial community in the rhizosphere and roots of Cyperus rotundus L. grown in a petroleum-contaminated soil.

Cyperus rotundus L. is a perennial herb which was found prevailing in an area in the northeast of Brazil previously contaminated with petroleum. In order to enlarge the knowledge of microorganism-plant interaction in phytoremediation, the bacterial community present in the rhizosphere and roots of C. rotundus was evaluated by culture-dependent and molecular approaches. PCR-DGGE analyses based on the 16S rRNA gene showed that the profiles of bulk soil, rhizosphere and root samples had a high degree of similarity. A complex community of alkane utilizing-bacteria and a variable nitrogen-fixing community were observed when the PCR-DGGE analyses were based on the genes alkB and nifH, respectively. In addition, two clone libraries were generated from the alkB fragments of bulk and rhizosphere soils. Statistical analyses showed that the libraries were different concerning the alkB population composition. Using culture-dependent techniques, 209 bacterial strains were isolated from the rhizosphere and rhizoplane/roots of C. rotundus. Dot blotting analysis showed that the DNA from 17 strains hybridized, simultaneously, with the alkB and nifH probes. After partial 16S rRNA gene sequencing, these strains were affiliated with the genera Bosea, Cupriavidus, Enterobacter, Gordonia, Mycoplana, Pandoraea, Pseudomonas, Rhizobium and Rhodococcus. They can be considered of great potential for phytoremediation in this tropical soil area.

Bioremediation potential of a tropical soil contaminated with a mixture of crude oil and production water.

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.

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

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.