Fig. 8. Various techniques for screening co-cultures for the activation of biosynthesis of secondary metabolites. (A) Using chemometric approaches (principal component analysis, heat maps, etc.) to determine how chemically similar or dissimilar the co-culture is to the monocultures allows for prioritizing fungal–fungal pairings. During chemometric analysis if a co-culture pairing demonstrates unique features, then that co-culture pairing can be further analysed. In contrast, if no unique features are observed, then that pairing will not be pursued for new chemical diversity. (B) Utilizing mass spectrometry to determine if any unique masses appear in the co-culture that are not present in either monoculture. The extract of both the monocultures and the co-culture can be analysed using mass spectrometry. If new masses appear in the co-culture that were absent in the monocultures, then those fungal–fungal pairings can be further pursued. In contrast, if no new signals arise, then the co-culture isn't inducing any new metabolites. (C) In situ analysis (e.g., droplet probe, MALDI, DESI, etc.) can be used to determine differential expression of metabolites by searching for unique masses in the co-culture that weren't present in the monocultures. Along the same lines as the mass spectrometry experiment, using in situ analysis can be a targeted way to determine new production from the co-culture compared to the monocultures. (D) Nuclear Magnetic Resonance (NMR) to determine if any unique signals appear that aren't present in either monoculture. The extract of both the monocultures and the co-culture undergoes NMR analysis. If new resonances appear in the spectrum of the co-culture that aren't present in either monoculture, then those fungal–fungal pairings can be further pursued. In contrast, if no new resonances arise, then the co-culture isn't inducing any new metabolites. (E) Using biological assays as a screening procedure allows for the identification of newly activated bioactive metabolites. If the biological assay of the co-culture shows an increase in activity in comparison to the monocultures, then the co-culture has activated the production of bioactive compounds. This can be used as a viable screener for the co-culture's ability to produce bioactive compounds; however, the caveat is that it is specific for a single type of bioassay.