Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudo-nitzschia multiseries

Axenic cultures of Pseudo-nitzschia multiseries (formerly Pseudonitzschia pungens f. multiseries) produce less domoic acid(DA) than the original bacteria-containing cultures. Bacterial strains isolated from two nonaxenic P. multiseries clones were reintroduced individually into cultures of three axenic P. multiseries strains. The bacteria did not substantially affect division rates or cell yields. However, they did cause a 2- to 95-fold enhancement of DA production (per cell basis) relative to the axenic culture, depending on the P. multiseries and bacterial strain used. Bacteria isolated from a nontoxic Chaetoceros sp. culture also enhanced DA per cell (by 115-fold), showing that it is not necessary for the bacteria to be isolated from a toxic culture in order to enhance toxin production. There was no evidence of intracellular bacteria in disrupted P. multiseries cells obtained from axenic cultures. Our results demonstrate an important, but nonessential, role of extracellular bacteria in DA production. Characterization of the bacterial strains using morphology, substrate utilization, and restriction fragment length polymorphism (RFLP) analyses clearly showed that we had isolated different species of bacteria from the various nonaxenic cultures. We conclude that not one but several bacterial species enhance DA production by P. multiseries.