Removal of extracellular material techniques and pitfalls

Extracellular material—specifically capsular material—has been strongly implicated in the flocculation of activated sludge and other mixed cultures. This study examines the steps involved to extract and collect this material, and suggests one suitable method which evolved after a study of numerous alternatives. Five steps are needed to obtain extracellular capsular material from the mixed culture which produced it. They are biomass concentration and removal of slime (washing); stripping of the capsule from the cell; precipitation of the stripped material from solution; collection of the precipitate; and an optional purification step. In the study reported herein, it was found that washing was essential to eliminate slime, as well as any dissolved salts which could contaminate the final product. Physical stripping by blending followed by centrifugation was more convenient than chemical stripping using K 2HPO 4; high-speed centrifugation alone did not result in the capsule being stripped from the cell. Precipitation was accomplished by adding the centrate to ethanol and refrigerating overnight. The precipitate was collected by filtering through tared glass-fibre filter paper. Further studies were done to assess whether blending would cause cell disruption (hence contamination by intracellular material), what level of repeatibility could be expected, and whether a single-pass blending step would remove all of the capsule. DNA measurements of the extract and of lysed cells showed that no significant disruption had been caused by blending. Duplicate tests indicated that repeatability of approx. 5% or better could be expected. Successive extractions showed that significant quantities of material (as much as 50% of the first extraction) could be removed by two further blending steps, but it is also likely that these extracts would contain higher amounts of intracellular material. Extracellular material is frequently referred to as “biopolymer”. However, because this material, having been extracted by methods as described above, has been shown to include organic monomers as well as inorganic components, an alternative description is suggested. This uses the precipitating agent (generally an alcohol) as the basis, and in the present study the term “ethanol insoluble material (eim)” has been used.