Supercritical fluid extraction of microbial phospholipid fatty acids from activated sludge
Introduction
Characterization of microbial communities is crucial to understanding environmental health. Compositions and functions of microbial community structure have been effectively used in bioremediation [1], [2], [3], [4], biomonitoring [5], [6], biological wastewater treatment [7], [8], [9], anaerobic digestion systems [10], [11], [12] and microbial taxonomy [13], [14].
Recent methodological advances in microbial ecology research are increasing our understanding of the composition and function of microbial communities in a wide variety of environmental samples. Many of the new methods extract the cellular constituents of microorganisms directly from the sample, eliminating the bias inherent in culture-based methods [15], [16].
One of the most commonly used culture-independent tools for investigating microbial populations in environmental research is the analysis of PLFA profiles. Phospholipids are major cell membrane constituents, and their fatty acyl side-chains vary in composition (i.e., length, alkyl-branches and number of double bonds) between eukaryotes and prokaryotes, as well as among many prokaryotic groups. Therefore, PLFAs are used as biomarkers to determine the presence and the abundance of specific microbial groups in their habitats. Since PLFAs are quickly degraded upon microbial death, they represent a ‘fingerprint’ of the viable microbial community and do not function as storage compounds [17], [18].
Analysis of PLFAs is traditionally performed by liquid organic solvent extraction (LSE) with a chloroform–methanol mixture [15], [19], [20]. The lipids are separated with silicic acid column chromatography into neutral lipids, glycolipids and phospholipids using chloroform, acetone and methanol as solvents. Fatty acids from the phospholipidic fraction are usually transformed into their less polar methyl ester derivatives (FAMEs) for analysis by gas chromatography. As a general standard procedure, this method has some disadvantages, such as the use of large quantities of hazardous solvents and being time consuming.
To overcome these problems, supercritical fluid extraction (SFE) has gained popularity as a common extraction technique in many areas because it offers a faster extraction method and lower solvent volume [21], [22]. Carbon dioxide is the preferred extraction fluid in SFE because it has relatively low critical values (31.1 °C and 7.4 MPa), is non-toxic, and it does not create environmental problems when used at the analytical scale.
In our previous study, extraction using scCO2 was successfully applied to determine the microbial ubiquinones and menaquinones from activated sludge [23], [24]. However, little research has been reported regarding the application of SFE for PLFA analysis. ScCO2 extraction has been used to extract fatty acids from whole bacterial cells by chemical derivatization [25], [26]. An integration system with sequential scCO2 extraction for the identification of lipid biomarker including PLFA has also been proposed elsewhere [27].
The objective of the present work is to study the suitability of SFE as an alternative method to extract microbial PLFAs from activated sludge. The extraction conditions were optimized by varying the following: extraction time, extraction temperature, extraction pressure, and modifier concentration. The effect of various modifiers on the total amount of extracted PLFA was also studied. The liquid organic solvent extraction method was used to evaluate the reliability of the SFE method.
Section snippets
Chemicals
All solvents were HPLC grade, and all other chemicals were analytical grade. Methanol, acetone, chloroform, ethanol and hexane, K2HPO4 and 1N HCl were purchased from Wako Co., Japan. Standards fatty acid methyl esters (Supelco 37-component FAME mix; Cat. No. 47885-U) and 0.5N Methanolic HCl (Supelco; Cat. No. 33095) were purchased from Sigma–Aldrich Co., Japan. The Sep-Pak Plus Silica cartridges were purchased from Waters Co., Japan. The Sep-Pak is a column, 10 mm in inner diameter and 20 mm in
Results and discussion
Activated sludge is a typical mixed culture of microorganisms [7]. The study of activated sludge microbial community structure can provide useful information to solve many problems in wastewater treatment plants operation [29]. Dried activated sludge samples with particles smaller than 500 μm was used in this study. Generally, the larger surface area of small particles results in an increase in the extraction efficiencies. In addition, excessive grinding of sample can severely impede the
Conclusions
In this work, an alternative method for the extraction of microbial PLFAs from activated sludge by using scCO2 extraction has been investigated. The experimental results have demonstrated that the application of scCO2 extraction to microbial PLFA analysis has the potential to drastically reduce the amount of solvent used and extraction time needed, and could simplify the procedure. Further studies are in progress to improve this method as an effective technique for analyzing microbial
Acknowledgement
This work was financially supported by a Grant-in-Aid for Scientific Research (code: B) from the Japan Society for the Promotion of Science (JSPS).
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