Use of the chemiluminescent probe lucigenin to monitor the production of the superoxide anion radical in a recombinant Aspergillus niger (B1-D)

Direct detection of intracellular superoxide anion radical (O-2(.-)) produc tion is of critical importance for investigating the responses of filamento us fungi to oxidative stress in bioprocesses. The purpose of this study is to establish a reliable method to monitor the O-2(.-) production within pel lets of Aspergillus niger. Addition of pure oxygen and the redox cycling ag ent paraquat to fungal pellet suspensions resulted in a considerable increa se in lucigenin-derived chemiluminescence (LDCL). In the presence of exogen ous superoxide dismutase (SOD), the LDCL of a disrupted cell solution was i nhibited. In contrast, with addition of diethyldithiocarbamate and sodium a zide, respectively, the inhibitors of Cu, Zn-SOD and Mn-SOD, an increased L DCL was observed. Further, as a probe, lucigenin can be absorbed and accumu lated in fungal pellet within a few minutes. Various pretreatments of the b ioreactor sample for the measurement of LDCL, were also investigated in the present study, and the use of intact pellets was adopted here rather than disrupting cells because the latter treatment led to difficulties in LDCL m easurement. These results show that lucigenin may be used as a convenient c hemiluminescent probe to monitor intracellular production of O-2(.-) in fil amentous fungi, and thus to follow changes in the level of this stressor wi thin fungi (C) 2001 John Wiley & Sons, Inc.