Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

Abstract

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.

Biogeography of bacterial communities in hot springs: a focus on the actinobacteria

“…Metagenomic DNA was extracted from sediments using the protocol described by Ellis et al [12] washed with 300 ml of 70% ethanol and air dried before resuspending in 50 ml of ultrapure water. Genomic DNA was extracted in triplicate, pooled and purified using PVPP spin columns [6].…”

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

“…After 6 weeks incubation, cells were collected from R2A and R2A/8 plates by adding 2 9 5 ml of sterile 0.9 % saline to the plate and suspending the colonies with a sterile plastic spreader (plate wash). The plate wash suspensions were collected into 15-ml sterile plastic tubes and homogenized by repeated pipetting (Ellis et al 2003).…”

Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches

“…There are numerous examples where culture-dependent methods were used to investigate the impacts of heavy metals, organic contaminants and herbicides on the diversity of the soil culturable bacterial community due both to the suppression of some bacterial groups and the enrichment of some contaminant-resistant bacterial taxa (Thompson et al 1998;Ellis et al 2001Ellis et al , 2003Ratcliff et al 2006). Ellis et al (2003) indicated that the readily culturable bacteria could be the largest, most active prokaryotes in a given sample and so provide a useful, rapid assessment of biological responses to heavy metal pollution. Thus, although culturedependent approaches of microbial community analysis are often criticized for their selectivity, it is perhaps this discrimination that makes them useful for determining the impact of anthropogenic activity.…”