title: Extraction of cyanobacterial endotoxin.
abstract: To simplify our efforts in acquiring toxicological information on endotoxins produced by cyanobacteria, a method development study was undertaken to identify relatively hazard-free and efficient procedures for their extraction. One article sourced and two novel methods were evaluated for their ability to extract lipopolysaccharides (LPSs) or endotoxins from cyanobacteria. The Limulus polyphemus amoebocyte lysate (LAL) assay was employed to compare the performance of a novel method utilizing a 1-butanol-water (HBW) solvent system to that of Westphal's (1965) phenol-water system (HPW) for the extraction of endotoxin from various cyanobacteria. The traditional HPW method extracted from 3- to 12-fold more endotoxin from six different cyanobacterial blooms and culture materials than did the novel HBW method. In direct contrast, the novel HBW method extracted ninefold more endotoxin from a non-microcystin producing Microcystis aeruginosa culture as compared to the HPW method. A solvent system utilizing N,N'-dimethylformamide-water (HDW) was compared to both the HPW and HBW methods for the extraction of endotoxin from natural samples of Anabaena circinalis, Microcystis flos-aquae, and a 1:1 mixture of Microcystis aeruginosa/Microcystisflos-aquae. The LAL activities of these extracts showed that the novel HDW method extracted two- and threefold more endotoxin from the Anabaena sample that did the HBW and HPW methods, respectively. The HDW method also extracted approximately 1.5-fold more endotoxin from the Microcystis flos-aquae sample as compared to both the HBW and HPW methods. On the other hand, the HBW method extracted 2- and 14-fold more endotoxin from the Microcystis flos-aquae/Microcystis aeruginosa mixture than did the HPW and HDW methods, respectively. Results of this study demonstrate that significant disparities exist between the physicochemical properties of the cell wall constituents not only of different cyanobacterial species but also of different strains of the same cyanobacterial species, as showing by the varying effectiveness of the solvent systems investigated. Therefore, a sole method cannot be regarded as universal and superior for the extraction of endotoxins from cyanobacteria. Nevertheless, the ability of the novel HBW and HDW methods to utilize easily handled organic solvents that are less hazardous than phenol render them attractive alternatives to the standard HPW method.
authors.affiliation: CRC for Water Quality and Treatment, Australian Water Quality Centre, Private Mail Bag 3, Salisbury, South Australia, 5108, Australia. john.papageorgiou@sawater.com.au
authors.forename: John
authors.initials: J
authors.lastname: Papageorgiou
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Linke
authors.forename: Con
authors.initials: C
authors.lastname: Kapralos
authors.forename: Brenton C
authors.initials: BC
authors.lastname: Nicholson
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Steffensen
mesh_terms: D001681:Biological Assay / Q000379:methods; D000458:Cyanobacteria / Q000737:chemistry*; D004731:Endotoxins / Q000302:isolation & purification*; D012997:Solvents / Q000737:chemistry; D013048:Specimen Handling
chemical_list: D004731:Endotoxins; D012997:Solvents