Supercritical Carbon Dioxide Extraction of Ubiquinones and Menaquinones From Activated Sludge
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Journal of Chromatography A, 1113 (2006) 14-19
JOURNAL OF CHROMATOGRAPHY A
www .elsevier.com!locate! chroma
Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge
Irvan a, b, Yoichi Atsuta a, Takashi Saeki a, Hiroyuki Daimon a,*, Koichi Fujie a
a Department of Ecological Engineering, Toyohashi University ofTechnology. Toyohashi 441-8580. Japan b Department of Chemical Engineering. University ofNorth Sumatra. JI. Alumni Kampus USU Medan 20155. Indonesia
Received 16 November 2005; received in revised form 24 January 2006; accepted 25 January 2006 Available online 10 February 2006
Abstract
Supercritical CO2 (scC02) extraction, with methanol as modifier, was applied to the determination of ubiqui nones and menaquinones in activated sludge. Four ubiquinones and 12 menaquinones species were identified based on retention time and UV spectrum in 0.1 g dried activated sludge. The optimum extraction conditions were at a pressure of 25 MPa, a temperature of 55 °C, and 10% (v/v) methanol for 15 min. At this condition, the concentrations of extracted ubiquinones and menaquinones were found to be 0.181 and 0.326/Lmollg-dry-cell, respectively. The results were comparable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Furthermore, the method was evaluated in term of repeatability, which resulted in an RSD of ~ 10%. The experimental results have demonstrated the technique to be simple, fast, and with less consumption of organic solvents. This work shows the potential application of supercritical CO2 extraction to microbial community analysis using quinone profile. © 2006 Elsevier B.Y. All rights reserved.
Keywords: Supercritical fluid extraction; Activated sludge; Quinone profile; Ubiquinones; Menaquinones
1. Introduction
Analysis of microbial community structure has become increasingly important nowadays. Microbial community structure plays a significant role in many fields such as biological wastewater treatment [1-3], microbial taxonomy [4-6], bioremediation [7,8] and environmental assessment [9,10]. Various methods, without the necessity of growing microorganisms in the laboratory, have been developed to describe microbial communities. Most of the methods are based on the characterization
of biomarkers, such as 16S~rDNA, total DNA, phospholipid
fatty acids, ergosterols, muramic acids and respiratory quinones [11-14]. Among these methods, the use of quinone profiles has an advantage in terms of quantification, simplicity, and reproducibility [15]. It makes this ~echnique a useful tool for the analysis of microbial population dynamics in mixed cultures [16].
• Corresponding author. Tel.: +81 53244 6910; fax : +8153244 6910. E-mail address: daimon@eco .tut.ac.jp (H. Daimon).
Microbial quinone exists in almost all microorganisms, and plays an important role as an electron carrier in the respiratory chain. In general, a species or genus of bacteria has only one dominant type of quinone. So, the quinone profile, which is usually represented as the molar ratio of each quinone type, should be specific for microbial community. There are two major groups of microbial quinones: the ubiquinones (l-methyl-2-isoprenyl3, 4-dimethoxy-parabenzoquinone) and the menaquinones (1isoprenyl-2-methyl-naphtho-quinone) [17]. The quinones are expressed in this manuscript in abbreviated form. UQ and MK represent ubiquinones and menaquinones, respectively. A hyphen separates the quinone type and the number of isoprenoid units. The number in parenthesis corresponds to the number of hydrogen atoms in the hydrogenation of double bonds in the side chain. For example, UQ-lO represents a ubiquinone with 10 isoprenoid units, and MK-9(H2) represents a menaquinone with 9 isoprenoid units. One of these units is hydrogenated with 2 hydrogen atoms.
In conventional solvent extraction methods, a mixture of chloroform and methanol is usually used to extract bacterial quinones from environmental samples [4]. This procedure yields
0021-9673/$ - see front matter © 2006 Elsevier B. V. All rights reserved. doi: 1O.1016/j.chroma.2006.01.112
Universitas Sumatera Utara
·f
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
·.
Available online at www.sciencedirect.com
8CIENCE@DIRECTO
Journal of Chromatography A, 1113 (2006) 14-19
JOURNAL OF CHROMATOGRAPHY A
www .elsevier.com!locate! chroma
Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge
Irvan a, b, Yoichi Atsuta a, Takashi Saeki a, Hiroyuki Daimon a,*, Koichi Fujie a
a Department of Ecological Engineering, Toyohashi University ofTechnology. Toyohashi 441-8580. Japan b Department of Chemical Engineering. University ofNorth Sumatra. JI. Alumni Kampus USU Medan 20155. Indonesia
Received 16 November 2005; received in revised form 24 January 2006; accepted 25 January 2006 Available online 10 February 2006
Abstract
Supercritical CO2 (scC02) extraction, with methanol as modifier, was applied to the determination of ubiqui nones and menaquinones in activated sludge. Four ubiquinones and 12 menaquinones species were identified based on retention time and UV spectrum in 0.1 g dried activated sludge. The optimum extraction conditions were at a pressure of 25 MPa, a temperature of 55 °C, and 10% (v/v) methanol for 15 min. At this condition, the concentrations of extracted ubiquinones and menaquinones were found to be 0.181 and 0.326/Lmollg-dry-cell, respectively. The results were comparable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Furthermore, the method was evaluated in term of repeatability, which resulted in an RSD of ~ 10%. The experimental results have demonstrated the technique to be simple, fast, and with less consumption of organic solvents. This work shows the potential application of supercritical CO2 extraction to microbial community analysis using quinone profile. © 2006 Elsevier B.Y. All rights reserved.
Keywords: Supercritical fluid extraction; Activated sludge; Quinone profile; Ubiquinones; Menaquinones
1. Introduction
Analysis of microbial community structure has become increasingly important nowadays. Microbial community structure plays a significant role in many fields such as biological wastewater treatment [1-3], microbial taxonomy [4-6], bioremediation [7,8] and environmental assessment [9,10]. Various methods, without the necessity of growing microorganisms in the laboratory, have been developed to describe microbial communities. Most of the methods are based on the characterization
of biomarkers, such as 16S~rDNA, total DNA, phospholipid
fatty acids, ergosterols, muramic acids and respiratory quinones [11-14]. Among these methods, the use of quinone profiles has an advantage in terms of quantification, simplicity, and reproducibility [15]. It makes this ~echnique a useful tool for the analysis of microbial population dynamics in mixed cultures [16].
• Corresponding author. Tel.: +81 53244 6910; fax : +8153244 6910. E-mail address: daimon@eco .tut.ac.jp (H. Daimon).
Microbial quinone exists in almost all microorganisms, and plays an important role as an electron carrier in the respiratory chain. In general, a species or genus of bacteria has only one dominant type of quinone. So, the quinone profile, which is usually represented as the molar ratio of each quinone type, should be specific for microbial community. There are two major groups of microbial quinones: the ubiquinones (l-methyl-2-isoprenyl3, 4-dimethoxy-parabenzoquinone) and the menaquinones (1isoprenyl-2-methyl-naphtho-quinone) [17]. The quinones are expressed in this manuscript in abbreviated form. UQ and MK represent ubiquinones and menaquinones, respectively. A hyphen separates the quinone type and the number of isoprenoid units. The number in parenthesis corresponds to the number of hydrogen atoms in the hydrogenation of double bonds in the side chain. For example, UQ-lO represents a ubiquinone with 10 isoprenoid units, and MK-9(H2) represents a menaquinone with 9 isoprenoid units. One of these units is hydrogenated with 2 hydrogen atoms.
In conventional solvent extraction methods, a mixture of chloroform and methanol is usually used to extract bacterial quinones from environmental samples [4]. This procedure yields
0021-9673/$ - see front matter © 2006 Elsevier B. V. All rights reserved. doi: 1O.1016/j.chroma.2006.01.112
Universitas Sumatera Utara
·f
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara