THE STUDY OF BACTERIA THAT METABOLIZE AROMATIC HYDROCARBONS BY STABLE ISOTOPE PROBING, SECONDARY ION MASS SPECTROMETRY, AND IN VITRO EXPERIMENTS WITH POLAROMONAS NAPHTHALENIVORANS CJ2
Stable isotope probing is a cultivation-independent technique that allows researchers to identify microorganisms that metabolize substrates that are labeled with stable isotopes. However, to understand the genetics and physiology of microbial processes, experiments using pure culture are often necessary. Polaromonas naphthalenivorans CJ2 was identified as a key member of the naphthalene-degrading community in coal tar-contaminated sediment, and was successfully isolated in pure culture. Using respirometry, metabolite detection by gas chromatography/mass spectrometry, and cell-free enzyme assays, strain CJ2 was shown to metabolize naphthalene via gentisate. Growth assays revealed that strain CJ2 is inhibited by naphthalene concentrations of 78 ?M (10 ppm) and higher. Despite being able to use naphthalene as a carbon and energy source, strain CJ2 must balance naphthalene utilization against two types of toxicity. Naphthalene directly inhibited growth, and the accumulation of putative naphthalene metabolites resulted in the loss of cell viability. Stable isotope probing of benzoic acid metabolizing bacteria in agricultural soil revealed the role of Burkholderia species, and cultivation efforts led to isolation of a representative of the benzoic acid-degrading population, Burkholderia sp. strain EBA09. Growth of the population represented by EBA09 in the field was demonstrated using MPN-PCR. The potential for dynamic secondary ion mass spectrometry (SIMS) ion microscopy to complement SIP studies by measuring 13C assimilation into individual bacterial cells grown on 13C-labeled organic compounds was explored. A clear relationship between mass 27 and 26 signals in cells grown in media containing varying proportions of 12C- to 13C-glucose was observed; a standard curve was generated to predict 13C-enrichment in unknown samples. Differences in 13C signals measured by SIMS were shown to be due to 13C assimilation into cell biomass. The application of SIMS ion microscopy to soil samples from a field experiment receiving 12C- or 13C-phenol revealed that 13C-labeled cells were detected in soil that was dosed a single time with 13C-phenol, and in soil that received 12 doses of 13C-phenol, 27% of the cells in the total community were more than 90% 13C-labeled.
Stable Isotope Probing; secondary ion mass spectrometry; Polaromonas naphthalenivorans; SIP; SIMS; naphthalene; benzoic acid; phenol
dissertation or thesis