Enrichment cultures are used to isolate organisms with degradative capabilities. The substrate to be tested is supplied as the sole carbon source in a medium to which mixed culture of microorganisms is added. Mixed culture source could be municipal sludge, soil, and river, or ocean water. Source of the microorganism is selected from the environment most likely contaminated with the substrate of interest. Only organisms with ability to degrade the substrate grow and become dominant population. These organisms are then isolated and purified. The purified microorganisms are then added back to a medium containing the substrate of interest as sole carbon source. Degradation of the substrate by these microorganisms is demonstrated generally using radiolabeled substrates. Identification of isolate also permits the result of such studies to be analyzed with other studies. These bacteria are deposited in the culture collection for use worldwide.
Since conditions in pure culture are different from those in the natural environment, the results obtained by pure culture studies may not be representative. It is common practice now to introduce the test chemicals at higher concentrations in samples collected from the environment, such as soil or water, and incubated under conditions similar to the environment.
It is also important to identify the metabolites or by-products resulting from the substrate biodegradation. For this purpose gas chromatography (GC) is used. GC is a sophisticated separation technique that requires low sample concentration. This technique is highly sensitive, accurate, and reproducible. The sample may be in solid, liquid, or gaseous phase as long as it is volatilized at the operating temperature of the instrument. The sample to be analyzed is injected along with the gas, which carries the sample along a column packed with inert particles coated with a liquid. The solutes in the sample are distributed between the liquid and gas phases according to the solubility of solute in the liquid. Solutes of low solubility move through the column at faster rates. As the band exits the column, they are recorded as peaks with retention time related to their partition coefficients. The peaks can be compared with the retention times of standard substrates. The solutes separated by GC can be analyzed via mass spectrometer (MS) to determine molecular structures of the compounds.
Use of radiolabeled compounds is another very sensitive technique to monitor biodegradation of substrates. In this method, biodegradation can be stopped and remaining quantity of the compound can be measured by counting the radioactive emission of the solution in liquid scintillation counter. Use of radiolabeled elements (e.g., 14C) is very useful to demonstrate whether biodegradation is complete or partial. In complete degradation (mineralization), CO2 is evolved. Mineralization of substrate results in accumulation of radioactive CO2, which can be quantified.
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