Full length ArticleMapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets
Introduction
Fungi continuously exist and interact with other fungi and microbes as a fundamental aspect of survival in harsh environments. Such symbiotic interactions shape fitness traits and create new adaptive techniques for microorganisms [1]. It has become an increasingly interesting topic to study, especially in the industrial biotechnology field, where co-culture conditions promote advantages such as increased product yield, quality control improvements and the potential to substitute cheaper substrates [2]. Co-cultures can also give rise to, but not limited to, production of new chemical substances from secondary metabolites [3], such as pharmaceuticals or active antibacterial compounds, improve the quality of existing products, including aroma and flavor substances for food and beverages [4,5], and allow simultaneous processes of complex multistep systems as in biofuel production [6].
This article focuses on the relationship of two fungi, Saccharomyces cerevisiae and Penicillium chrysogenum, which are known to form spontaneously hollow spheres and remain viable while attached to one another, creating a yeast immobilization system termed’ yeast biocapsules’ [7]. These form in aseptic synthetic liquid media deprived of nutrients and with gluconic acid as the only carbon source. In prior investigations, fusion of cell walls of S. cerevisiae yeast and P. chrysogenum hyphae has been observed in transmission electron microscope images [8]. Cross sections show the plasma membranes of the two organisms in close contact. Circular organelles within the yeast cytoplasm, thought to be vesicular structures, conglomerate at the point of junction [9]. These observations raised speculations that the yeast and filamentous fungus (ff) were closely attached to exchange metabolites for the benefit of one or both partners.
Applications of biocapsules have been well studied including advantages in the production of fermentation products such as wine and bioethanol [[9], [10], [11], [12], [13]]. However, little is known of the molecular transfers occurring between the yeast and ff, which could serve as valuable information to improve the application of biocapsules or to develop them as a new sector of biotechnology. Co-culture conditions are complex and challenging to study due to numerous changing variables contributed by both organisms. Current advances in metabolomic research have allowed a better understanding of these complex consortia in greater depth [14,15]. Here, an intracellular metabolomic approach is taken, as well as tracking other parameters (gluconic acid consumption, biomass growth, immobilization yield and primary amino acid release), to gain further insight into the responses of yeast and ff in biocapsules, through analysis of the evolution of metabolite changes by comparing the co-culture with single culture conditions of each.
Section snippets
Microorganisms and growth media
Flor yeast strain S. cerevisiae G1 (ATCC® MYA-2451™) and ff strain P. chrysogenum H3 were used. The two strains were selected after screening for yeast-fungus co-adhesion where they produced the most consistent, stable biocapsules suitable for use in fermentation processes [7,16]. The flor yeast characteristic of forming biofilm has been significantly correlated to development of consistent biocapsules with high immobilization yields. The yeast cells were pre-grown on YPD-agar (1 % yeast
Gluconic acid consumption
Gluconic acid was consumed most rapidly in the biocapsule condition, where by day 3 it was no longer detectable (Fig. 1). It was also not detectable in ff by day 3, but was not consumed as rapidly during the first day of incubation compared to biocapsules. Yeast cells consumed the gluconic acid slowly and in small quantity (ca. 1.5 g/L by the end of day 6). It can be speculated that the rapid decrease in gluconic acid in biocapsule condition at day 1 was due to the combined consumption by yeast
Discussion
The results of this study show that the forced co-cultivation condition of the yeast and filamentous fungus in a medium with low nutrients and lacking fermentable carbon sources impacted on the growth conditions of both organisms. The untargeted metabolomic fingerprinting differentiated between each condition, indicating that the co-culture induces a unique endometabolome profile which is significantly different than either of the pure culture conditions.
Conclusion
This work has defined the responses and metabolomic changes that occurred in yeast and ff in co-culture conditions where the two organisms adhere to each other. Both organisms remain viable after complete biocapsule formation, but it appears that the ff are hindered from growing in biomass under co-cultivation conditions while yeast cells increase attachment to the hyphae. In Y-FC, there was an accumulation of toxic compounds synthesized by the ff, as well as a high accumulation of fructose, a
Funding
This study was supported by the XXIII Programa Propio de Fomento de la Investigación 2018 (MOD. 4.2. SINERGIAS, Ref. XXIII PP Mod. 4.2) from the University of Córdoba (Spain) and the Spanish Fulbright commission for granting a Predoctoral Research Fulbright Scholarship at the University of California, Davis, to Jaime Moreno-García.
Declaration of Competing Interest
The authors declare no conflict of interest.
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