Application of Modified Supercritical Carbon Dioxide Extraction to Microbial Quinone Analysis
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Appl Microbiol Biotechnol (2006) 69: 506-s'b\} DOl 10. 1007/s00253-005-0008-8
BIOTECHNOLOCHCAL PRODUCTS AND PROCESS ENGINEERING
Irvan . Udin Hasanudin . Muhammad Faisal . Hiroyuki Daimon . Koichi Fujie
Application of modified supercritical carbon dioxide extraction to microbial quinone analysis
Received: 21 February 2005 / Revised: 13 April 2005 / Accepted : IS April 2005 / Published online: 19 July 2005 Ii') Springer-Verlag 2005
Abstract Supercritical carbon dioxide (scC02) was applied to extract microqial quinones from activated sludge. Identification and analysis was then performed using highperformance liquid chromatography (HPLC) equipped with ultraviolet-visible (UV-Vis) detector and photodiode an'ay detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scC02 were similar to that of the conventional method (organic solvent extraction).
Irvan . U. Hasanudin . M. Faisal . H. Daimon (Id ) . K. Fujie Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, 441-8580, Japan e-mail : daimon@eco.tut.ac.jp Tel.: +81-532-446910 Fax: +81-532-446910
Irvan Department of Chemical Engineering, University of North Sumatra. JI. Alumni Kampus USU, Medan, 20 ISS, Indonesia
U. Hasanudin Department of Agroindustrial Technology, . University of Lampung, JI. Sumantri Brojonegoro I, Kampus Unila, Bandar Lampung, 35145, Indonesia
M. Faisal Department of Chemical Engineering, Syiah Kuala University, JI. Syech Abdul Rauf, 23111 NAD. Indonesia
1.~
Introduction
Microbial community stmcture is one of the important factors controlling the pollutant-degrading capacity of ecosystems. The capacity of an ecosystem to degrade organic compounds and its response to the changes in environmental conditions depend not only on the total popUlation of microorganisms present, but also on the microbial community stmcture of that system (Hu et al. I999a). Microbial population dynamics can be analyzed using quinone profiles. Nowadays, the technique of using quinone profiles has gained increased recognition as a simple and useful tool to analyze microbial population dynamics in mixed cultures (Hu et al. 2001a; Hedrick and White 1986). Quinones can be found in almost all bacteria. In general, one species or genus of bacteria has only one dominant type of quinone (Hess et al. 1979; Collins and Jones 1981). For that reason, quinone profile, which is usually represented as the mole fraction of each quinone type, should be specific for a microbial community. Changes in a microbial community in a mixed culture of microbes could be effectively quantified by using the quinone profiles (Hiraishi 1988). Quinone species are classified into two major groups: ubiquinones (1methyl-2-isoprenyl-3, 4-dimethoxyparabenzoquinone) and menaquinones (l-isopreny1-2-methyl-naphtho-quinone) (Collins and Jones 1981).
In the conventional method, analysis of quinones is perfonned by organic solvent extraction (direct extraction) with chloroform-methanol mixture (Collins and Jones 1981). Samples containing quinones are separated into a complex mixture oflipid and impurities. The impurities are removed by column chromatography, while microbial quinones are separated by thin layer chromatography (TLC). The compositions of purified fractions of ubiquinones and menaquinones are then analyzed by HPLC. Because satisfactory results could not be obtained using TLC, a solid-phase extraction method using Sep-Pak Plus Silica was employed for the purification and separation of quinones (Hu et at. 1999b). However, the conventional method has still some disadvantages: time-consuming, tedious and the use oflarge quantities ofvarious organic solvents, which have detrimen-
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Appl Microbiol Biotechnol (2006) 69: 506-s'b\} DOl 10. 1007/s00253-005-0008-8
BIOTECHNOLOCHCAL PRODUCTS AND PROCESS ENGINEERING
Irvan . Udin Hasanudin . Muhammad Faisal . Hiroyuki Daimon . Koichi Fujie
Application of modified supercritical carbon dioxide extraction to microbial quinone analysis
Received: 21 February 2005 / Revised: 13 April 2005 / Accepted : IS April 2005 / Published online: 19 July 2005 Ii') Springer-Verlag 2005
Abstract Supercritical carbon dioxide (scC02) was applied to extract microqial quinones from activated sludge. Identification and analysis was then performed using highperformance liquid chromatography (HPLC) equipped with ultraviolet-visible (UV-Vis) detector and photodiode an'ay detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scC02 were similar to that of the conventional method (organic solvent extraction).
Irvan . U. Hasanudin . M. Faisal . H. Daimon (Id ) . K. Fujie Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, 441-8580, Japan e-mail : daimon@eco.tut.ac.jp Tel.: +81-532-446910 Fax: +81-532-446910
Irvan Department of Chemical Engineering, University of North Sumatra. JI. Alumni Kampus USU, Medan, 20 ISS, Indonesia
U. Hasanudin Department of Agroindustrial Technology, . University of Lampung, JI. Sumantri Brojonegoro I, Kampus Unila, Bandar Lampung, 35145, Indonesia
M. Faisal Department of Chemical Engineering, Syiah Kuala University, JI. Syech Abdul Rauf, 23111 NAD. Indonesia
1.~
Introduction
Microbial community stmcture is one of the important factors controlling the pollutant-degrading capacity of ecosystems. The capacity of an ecosystem to degrade organic compounds and its response to the changes in environmental conditions depend not only on the total popUlation of microorganisms present, but also on the microbial community stmcture of that system (Hu et al. I999a). Microbial population dynamics can be analyzed using quinone profiles. Nowadays, the technique of using quinone profiles has gained increased recognition as a simple and useful tool to analyze microbial population dynamics in mixed cultures (Hu et al. 2001a; Hedrick and White 1986). Quinones can be found in almost all bacteria. In general, one species or genus of bacteria has only one dominant type of quinone (Hess et al. 1979; Collins and Jones 1981). For that reason, quinone profile, which is usually represented as the mole fraction of each quinone type, should be specific for a microbial community. Changes in a microbial community in a mixed culture of microbes could be effectively quantified by using the quinone profiles (Hiraishi 1988). Quinone species are classified into two major groups: ubiquinones (1methyl-2-isoprenyl-3, 4-dimethoxyparabenzoquinone) and menaquinones (l-isopreny1-2-methyl-naphtho-quinone) (Collins and Jones 1981).
In the conventional method, analysis of quinones is perfonned by organic solvent extraction (direct extraction) with chloroform-methanol mixture (Collins and Jones 1981). Samples containing quinones are separated into a complex mixture oflipid and impurities. The impurities are removed by column chromatography, while microbial quinones are separated by thin layer chromatography (TLC). The compositions of purified fractions of ubiquinones and menaquinones are then analyzed by HPLC. Because satisfactory results could not be obtained using TLC, a solid-phase extraction method using Sep-Pak Plus Silica was employed for the purification and separation of quinones (Hu et at. 1999b). However, the conventional method has still some disadvantages: time-consuming, tedious and the use oflarge quantities ofvarious organic solvents, which have detrimen-
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara
Universitas Sumatera Utara