To promote the use of sewage sludge on farmland, a flocculant compatible with agricultural application is required. In general, an inorganic flocculant such as aluminium sulfate is used at large sewage disposal plants. However, there is concern that aluminium accumulates when it is returned to farmland, and therefore a biodegradable rather than an inorganic flocculant is required. To obtain biodegradable flocculants produced by microorganisms, the latter were isolated from soils and sewage sludges, and the flocculation activity of the culture broths was evaluated. Here, real sewage sludge was used for the evaluation because practical use was regarded as an important consideration. As a result, two strains, V13 and RK14, were selected and identified as Bacillus megaterium and Bacillus licheniformis, respectively. It was also found that flocculation was caused by poly-gamma-glutamic acid (gamma-PGA). gamma-PGA from RK14 cells had considerably higher molecular weight than commercial gamma-PGA, and the enantiomeric composition differed from that of gamma-PGA from other B.licheniformis. To obtain mutants with high gamma-PGA productivity, RK14 cells were treated with ethyl methanesulfonate, and a mutant (RK14-46) was selected. The culture medium suitable for gamma-PGA production by RK14-46 cells was totally different from that by RK-14 cells. The gamma-PGA production reached 21.1 g/L when RK14-46 cells were cultured in the optimum semisynthetic medium at 30 degrees C for 2 d. Furthermore, the combination of gamma-PGA obtained from RK14-46 cells and chitosan, a cationic flocculant, was very effective for flocculation of real sewage sludge.