Detecting Pseudomonas Sp. Strain ADP in An Atrazine Enriched Environment With Raman Spectroscopy

Raman spectroscopy is a technique that is seeing wider application for the analysis of biological samples because it offers the potential to distinguish cells in a non-destructive manner. Through measurement of vibrational, rotational, and other low-frequency modes in molecules, Raman spectroscopy enables rapid detection of chemical bond changes. Using confocal Raman microscopy, efforts are made to obtain three dimensional distributions of substances with high spatial resolution. For the purposes of this research, cellular and chemical changes occurring during the metabolic degradation of an environmental pollutant are traced with Raman spectroscopy. To allow for immobilization of contaminant, improved cell life, and increased horizontal gene transfer, organisms are grown as biofilms and examined with 532 and 785nm excitation lasers.  Proposed hypotheses are: 1) Raman scattering can be used to identify and distinguish bacteria in the free cell state and in a biofilm and 2) Raman scattering can be used to evaluate the concentration of the substrate and metabolites in flow systems.  

To create a model system Pseudomonas sp. strain ADP is grown in a nutrient limited medium with atrazine as its sole nitrogen source. Pseudomonas sp. strain ADP mineralizes atrazine through a series of hydrolytic reactions to carbon dioxide and ammonia. Along the degradation pathway five intermediates are produced: hydroxyatrazine, N-isopropylammelide, cyanuric acid, biuret, and allophanate. The accumulation and disappearance of these intermediates depend on the expression of atrazine pathway genes, which may be differentially expressed from the large atrazine degradation plasmid retained within the microorganism. For Raman analysis, microbial biofilms are grown in shake flasks and flow reactors under varying shear stress to allow for selection of optimal residence time required for cellular adhesion to atrazine.

Raman analysis of cells as well as atrazine and metabolites are being used to characterize the active biofilm. Success of this research will contribute to development of non-invasive techniques to examine both internal composition and spatial distribution of compounds present in biofilms. This will also lead to a firmer grasp of scientific fundamentals associated with pollutant degradation by biofilms, to improved remediation applications, and ultimately, to reduced pollutant-associated illness.  Gaining insight to conditions required to create systems conducive for efficient transformation by biofilm may have many useful applications environmentally, industrially, and medically.