Supercritical Fluid Extraction of Bacterial and Archaeal Lipid Biomarkers from Anaerobically Digested Sludge

Abstract

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile.

Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications

“…For improved SFE polarity and solvent strength, a modifier such as 10% MeOH was added to the supercritical solvent, as the polarity of CO 2 alone is not high enough for PLFA separation from sludge. It was evident that optimization of the SFE conditions was needed for a specific matrix (Hanif et al 2012). SFE has also been applied on activated sludge (Hanif et al 2010), bacterial cultures (Gharaibeh and Voorhees 1996) and archaeal cultures (Hedrick et al 1991).…”

“…This ionization leads to the increased loss of phospholipids into the aqueous phase, inducing a lower lipid yield in the organic phase. Other studies, utilizing buffered solvent systems for microbial lipid extraction, have been conducted on macroalgae (Kumari et al 2011); soil (Kehrmeyer et al 1996;G omez-Brand on et al 2008G omez-Brand on et al , 2010Chowdhury and Dick 2012;Ayari et al 2013); activated sludge (Conrad et al 2003;Hanif et al 2010Hanif et al , 2012; estuarine sediments (Guckert et al 1985), biofilms (Vinten et al 2011;Liu et al 2012) and pure microbial cultures (Macnaughton et al 1997). Consistent with the fact that microbial mats may contain Archaea, the use of 1% trichloroacetic acid instead of H 2 O, has been shown to successfully extract archaeal lipids from these matrices (Nishihara and Koga 1987;Allen et al 2010).…”

“…CO 2 has low critical values (31Á1°C and 7Á4 MPa), is nontoxic and does not give rise to environmental and health risks (Hanif et al 2010). Hanif et al (2012) used supercritical CO 2 extraction to simultaneously extract microbial respiratory quinones, PLFAs and phospholipid ether lipids from anaerobically digested sludge. For improved SFE polarity and solvent strength, a modifier such as 10% MeOH was added to the supercritical solvent, as the polarity of CO 2 alone is not high enough for PLFA separation from sludge.…”

Methods for quantification of growth and productivity in anaerobic microbiology and biotechnology

“…After performing a supercritical fluid extraction (SFE), bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge can be analyzed. Bacterial RQ were determined using UPLC (Hanif et al 2012 ). To identify and quantify liquid components in microbial cultures, mass spectrometry (MS) could be coupled to LC or HPLC.…”

Aplikasi Teknologi Hijau Untuk Pemantauan Kualitas Lingkungan Berbasis Struktur Komunitas Mikroba

“…[13]. Aplikasi EFS untuk mengekstrak mikroba lipid dapat memberikan efisiensi ekstraksi yang lebih tinggi [6,5,4].Karbon dioksida lebih umum digunakan sebagai fluida EFS karena memiliki nilai temperatur dan tekanan kritis yang relatif lebih rendah yaitu 31.1 °C dan 7.4 MPa.…”