Superfund Research Program
Ecological Genetics of PAH- and Metal-Transforming Bacteria
Project Leader: Brian K. Kinkle
Grant Number: P42ES004908
Funding Period: 2001 - 2006
- Project Summary
Final Progress Reports
The overall goal of this project is to examine the structure and function of microbial populations present in contaminated soils that are responsible for the transformation of toxic organic and heavy metals contaminants during bioremediation efforts. Dr. Kinkle’s experimental approach emphasizes molecular methods that focus on uncultured microbial strains that are active in situ, or in actual contaminated soil environments.
Dr. Kinkle’s lab has described the development of a culture-based biochip device for rapid detection of mycobacteria in environmental samples. Individual biochips rely upon the unique paraffinophilic nature of mycobacteria to rapidly and selectively adhere to the surface of the device. They used prototype biochips to experimentally demonstrate the concept of rapid and selective detection of mycobacteria by testing pure cultures and using epifluorescence microscopy to visualize microorganisms on the surface. As an alternative, rapid approach for identifying the biomass on the biochip surface, Dr. Kinkle’s lab used microwaves in the 10 to 26 GHz frequency range. The results of this study indicate that different microorganisms are responsible for specific shifts in resonance frequencies of a microwave cavity. By combing the semi-selective paraffin surface of the biochip with the microorganism-specific response to the microwaves, they have developed an improved analytical system with the potential to rapidly identify and enumerate mycobacteria in environmental samples in as little as 2 h.
Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.