Aust. ~7Mal: ~rihwater Res., 1994, 45, 863-8 Separation of Forms of Microcystis from Anabaena in Mixed Populations by the Application of Pressure Justin D. BrookesA, George G. GanfA and Michael D. BurchB ADepartment of Botany, The University of Adelaide, SA 5005, Australia. BAustralian Centre for Water Quality Research, PMB Salisbury, SA 5108, Australia. Abstract Critical-pressure distributions of gas vesicles in Anabaena circinalis, Microcystis aeruginosa f. aeruginosa and M. a. f. flos-aquae were determined for suspensions both in hypertonic sucrose solutions and in reservoir water. The differences between the critical and apparent critical pressures of gas vesicles suggested that differential pressurization could be used to separate these taxa. Subsequent experiments successfully separated (>90%) the two formae of Microcystis by the application of 500 kPa and M. a. f. aeruginosa from A, circinalis by the application of 300 kPa. This technique has the potential to provide sufficiently pure material to distinguish the relative toxicity of the two formae of Microcystis in the presence of a neurotoxic A . circinalis. Introduction Microcystis aeruginosa Kiitz. emend. Elenkin is a freshwater cyanobacterium that commonly forms blooms. Reynolds et al. (1981) described six morphological categories and nine stati, all belonging to M, a. f. aeruginosa. Two of the most prominent stati are the clathrate and quasi- spherical forms (Reynolds et al. 1981: plate 1, figs. 5, 6 and 9) that correspond to the descriptions of Microcystis aeruginosa formae aeruginosa and $08-aquae by Kondrat'eva (1968) . These stati coexist in Mt Bold Reservoir, South Australia, alongside another cyanobacterium, Anabaena circinalis (Oliver and Ganf 1988; Ganf et al. 1989). Both species are known toxin producers-Microcystis produces hepatotoxins and Anabaena a neurotoxin (Falconer 1989)- but it is uncertain whether all forms of Microcystis are equally toxic (Kruger et al. 1981). Although hepato- and neurotoxins can be distinguished by high-performance liquid chromatography (HPLC), the commonly used mouse bioassay is unable to differentiate between the two when both are present, because the neurotoxin takes effect before liver damage is apparent. Therefore, it would be convenient if there were a simple method for separating both forms of Microcystis and Anabaena. Gas vesicles are gas-filled structures found in cyanobacteria (Walsby 1975), and previous workers have shown a differential strength (critical pressure) of gas vesicles between genera of bacteria and cyanobacteria (Walsby 1972). For the common nuisance species such as Anabaena flos-aquae, Aphanizomenon flos-aquae, Microcystis aer~iginosa and Oscillatoria agardhii, critical pressures range between 500 and 900 kPa (Utkilen et al. 1985; Kromkamp et al. 1986; Konopka et al. 1987; Kinsman et al. 1991). The coexistence of Microcystis aeruginosa and Anabaena circinalis in Mt Bold Reservoir has hampered efforts to determine the problem taxa when water quality is compromised. This paper demonstrates that differential critical pressure can be exploited to separate species and/or forms of cyanobacteria for further biochemical analysis. The technique presented requires only the application of pressure, is inexpensive, and is not dependent on special equipment as seen in other separation systems (e.g. flow cytometry or density-gradient centrifugation).