Impact of Yeast Flocculation and Biofilm Formation on Yeast-Fungus Coadhesion in a Novel Immobilization System

Abstract

A novel method of yeast immobilization, called "biocapsules", has been developed in which cells of the yeast Saccharomyces cerevisiae become attached to the hyphae of the fungus Penicillium chrysogenum, remaining adhered following loss of viability of this fungus. Yeast immobilization facilitates higher cell densities than traditional fermentation methods, improves yield, and allows the reuse of the biocatalyst. Yeast cells may adhere to each other via specific cell-surface molecular interactions (flocculation) or may attach to surfaces (biofilm formation), and the roles of these two distinct attachment mechanisms in biocapsule formation is unknown. To elucidate the influence of biofilm formation versus flocculation on the yeast-fungus coimmobilization, a screening of selected strains from the Viticulture and Enology Department collection at the University of California, Davis, was carried out, and their ability to flocculate and form biofilm was quantified. Eighteen yeast strains capable of flocculation and biofilm formation were identified in this screening. Strains displaying differential capabilities in flocculation or biofilm formation and two control strains were further evaluated for their ability to specifically immobilize with P. chrysogenum. Seven strains exhibiting different patterns of flocculation and biofilm formation were identified. Biofilm-forming yeast strains displayed higher rates of immobilization with P. chrysogenum and formed more consistent biocapsules. In contrast, strains able to flocculate developed smaller, inconsistent biocapsules. Although the size and number of biocapsules formed varied by yeast strain, the total mass of biocapsules generated was similar for all strains. These results shed light on parameters that influence yeast-fungus coimmobilization, which may lead to an improvement of biocapsule consistency and advance the field of application for this new immobilization system.

FLO1, FLO5 and FLO11 Flocculation Gene Expression Impacts Saccharomyces cerevisiae Attachment to Penicillium chrysogenum in a Co-immobilization Technique

“…Biocapsule number and diameter are indistinguishable for FLO11 when expressed from either the ADH2 or HSP30 promoters, but the level of attachment of the cells varies dramatically (Figure 1C ), suggesting that strain parameters but not the physical action of attachment dictate biocapsule number and diameter. Moreno-García et al (2018b) supports this result with an earlier experiment and found that mass and volume of yeast biocapsules are highly strain dependent.…”

“…The ability of Flo proteins to be able to adhere to neighboring cells or other substrates may also explain the trend of large size but few in number of biocapsules (Figures 4 – 6 ). This linear regression matches with that of a previous study by Moreno-García et al (2018b) , where biocapsules made with different wild type yeast strains displayed large-few or small-abundant trends. Overexpressed FLO11 strains resulted in the largest diameter biocapsules perhaps because Flo11p allows for attachment with a longer retention and more permanency compared to Flo1p and Flo5p which are involved in flocculation only and no other FLO11 phenotypes, where attachment is easily broken by intense agitation.…”

“…It may be that in the case of the biocapsules, hydrophobic forces of Flo11p causes yeast cells to repulse from the liquid media and attract instead to the hydrophobic fungal spores and/or hyphae (cell-substrate adhesion) and then form biofilm within the hyphae matrix leading to stronger attachment ( Zhang and Zhang, 2016 ). Consistent with this observation, in a previous study ( Moreno-García et al, 2018b ), yeasts that were naturally able to form biofilm also showed higher rates of immobilization in biocapsules when compared with yeasts lacking this ability. Moreover, FLO11 can induce haploid invasive growth ( Lambrechts et al, 1996 ; Harashima and Heitman, 2002 ; Kuchin et al, 2002 ).…”

“…Immobilized yeast cells were quantified by taking ten biocapsules per flask and “disrupting” biocapsules with NaCl (100 mM), grinding them with a mortar and pestle for 2 min, and vortexing for 20 s in order to separate yeast cells from fungal hyphae. To clarify the sample from the fungus hyphae so that the yeast cells could be quantified, several successive differential filtrations were performed following Moreno-García et al (2018b) . Yeast cells were quantified by analyzing the filtered samples under a microscope and counting cells on a Hemocytometer at 40× objective and finally the number was normalized to the total number of biocapsules in each replicate.…”

New insights on yeast and filamentous fungus adhesion in a natural co-immobilization system: proposed advances and applications in wine industry

“…Even after complete fermentation, the biocapsules maintain their integrity which makes possible to reuse them for subsequent fermentations (Peinado et al 2006). Further, the biocapsules have been investigated to have high immobilization efficiency up to 84% yeast cell immobilized (Moreno-García et al 2018b) and unlike most immobilization methods, yeast biocapsules can prevent loss of cells from the carrier because of potential attachment of any newly formed daughter cells using the same natural process as the attached parental cells (Moreno-García et al 2018c). What determines the specific mechanism of attachment is still in question, but studies have shown that different yeast strains affect immobilization ability.…”