(804f) 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.