High-yield citric acid production by the filamentous Ascomycete fungus Aspergillus niger requires a combination of extreme nutritional conditions, of which maintaining a low manganese (II) ion concentration (<5 μg L−1) is a key feature. Technical-scale production of citric acid predominantly uses stainless-steel tank fermenters, but glass bioreactors used for strain improvement and manufacturing process development also contain stainless steel components, in which manganese is an essential alloying element. We show here that during citric acid fermentations manganese (II) ions were leaching from the bioreactor into the growth media, resulting in altered fungal physiology and morphology, and significant reduction of citric acid yields. The leaching of manganese (II) ions was dependent on the fermentation time, the acidity of the culture broth and the sterilization protocol applied. Manganese (II) ion leaching was partially mitigated by electrochemical polishing of stainless steel components of the bioreactor. High concentrations of manganese (II) ions during early cultivation led to a reduction in citric acid yield. However, the effect of manganese (II) ions on the reduction of citric acid yield diminished towards the second half of the fermentation. Since maintaining low concentrations of manganese (II) ions is costly, the results of this study can potentially be used to modify protocols to reduce the cost of citric acid production.

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species