Research noteFatty acid methyl ester (FAME) analysis for monitoring Nocardia levels in activated sludge
Introduction
Proliferation of Nocardia amarae cells in activated sludge has often been associated with nuisance foaming incidents that lead to the deterioration of effluent quality, increased time for plant maintenance, hazardous working conditions, and severe anaerobic digester operating problems (Pitt and Jenkins, 1990). Several researchers have successfully used Nocardia filament counting to evaluate various Nocardia control techniques in both bench-scale (Cha et al., 1992; Pagilla et al., 1996) and full-scale (Pitt and Jenkins, 1990) studies. Although the filament counting technique is straightforward and provides meaningful quantitative information on Nocardia levels in mixed liquor, it is time-consuming and labor intensive. In addition, the results are often influenced by Gram-staining preparation and the ability of the investigator to recognize Gram-stained Nocardia cells, especially with the presence of non-Nocardia filaments in activated sludge. This potential error may be compounded for Nocardia populations subjected to stress in an activated sludge process because, under such conditions, Nocardia cells are either weakly stained or completely lose staining characteristics (Pitt and Jenkins, 1990; Cha et al., 1992).
Analysis of fatty acid methyl esters (FAMEs) derived from cellular membrane lipids presents exciting possibilities for monitoring and quantifying the growth of Nocardia and possibly other organisms in activated sludge. All cellular organisms possess membranes composed largely of phospholipids. The specific phospholipids and their abundance depend on the particular organism and are sufficiently distinctive for the identification of a range of pure cultures (Lechevalier, 1977; Sasser, 1990). By extension, the technology has the ability to provide the relative abundance of the various fatty acids associated with Nocardia cells within a mixed liquor sample. The overall goal of this study was to evaluate the ability of FAME technology to selectively quantify Nocardia levels in the mixed liquor samples. Specifically, in order to distinguish Nocardia cells in activated sludge, fatty acids associated with N. amarae were determined and compared to fatty acid profiles of non-foaming activated sludge. From this information, signature fatty acids unique to Nocardia were identified. In addition, FAME profiles for mixed liquor samples spiked with various levels of Nocardia were generated to determine whether Nocardia levels can be quantitatively monitored by FAME analysis.
Section snippets
Materials and methods
The pure culture of Nocardia amarae used was originally isolated at the University of California, Berkeley, CA. Nocardia cells were grown in a 4-l batch reactor containing a buffered mineral salt medium with 2300 mg/l sodium acetate as the sole carbon source. Pure oxygen was provided in the headspace to deliver the required oxygen to the cells by surface aeration. Cells were harvested from batch reactors at day 6 which corresponded to the stationary phase of growth (Kim, 1997). The cells were
Results and discussions
The gas chromatograms of fatty acids released from pure Nocardia cells and an activated sludge sample are compared in Fig. 1. The profile for the activated sludge is relatively complex as it represents the combined fatty acids associated with all of the microbial types present in the sample (Fig. 1b). In contrast, only seven fatty acid peaks were observed for N. amarae cells grown in pure culture (Fig. 1a). Two of these seven peaks appeared to be unique to Nocardia when compared to the FAME
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