Gravity-Driven Adaptive Evolution of an Industrial Brewer’s Yeast Strain towards a Snowflake Phenotype in a 3D-Printed Mini Tower Fermentor

Andreas Conjaerts,Ronnie Willaert

doi:10.3390/fermentation3010004

Andreas Conjaerts, Ronnie Willaert

Open Access

https://doi.org/10.3390/fermentation3010004

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Journal: Fermentation	Publication Date: Jan 5, 2017
Citations: 10	License type: CC BY 4.0

Affiliation: Ghent University, Vrije Universiteit Brussel

Abstract

We designed a mini tower fermentor that is suitable to perform adaptive laboratory evolution (ALE) with gravity imposed as selective pressure, and suitable to evolve a weak flocculating industrial brewers’ strain towards a strain with a more extended aggregation phenotype. This phenotype is of particular interest in the brewing industry, since it simplifies yeast removal at the end of the fermentation, and many industrial strains are still not sufficiently flocculent. The flow of particles (yeast cells and flocs) was simulated, and the theoretical retainment advantage of aggregating cells over single cells in the tower fermentor was demonstrated. A desktop stereolithography (SLA) printer was used to construct the mini reactor from transparent methacrylic acid esters resin. The printed structures were biocompatible for yeast growth, and could be sterilised by autoclaving. The flexibility of 3D printing allowed the design to be optimized quickly. During the ALE experiment, yeast flocs were observed within two weeks after the start of the continuous cultivation. The flocs showed a “snowflake” morphology, and were not the result of flocculin interactions, but probably the result of (a) mutation(s) in gene(s) that are involved in the mother/daughter separation process.

Highlights

The use of evolutionary methods is a more “natural” approach to enhance the attributes of microorganisms, in contrast to genetic modification which has so far precluded its commercial use due to the low consumer tolerance for genetically modified organisms [1,2]
Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion [5]
We demonstrate that SLA 3D printing can be used to construct the mini tower fermentor, and that the design is suitable to obtain a yeast cell aggregation

Summary

Introduction

The use of evolutionary methods is a more “natural” approach to enhance the attributes of microorganisms, in contrast to genetic modification which has so far precluded its commercial use due to the low consumer tolerance for genetically modified organisms [1,2]. Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion [5]. A number of investigations have demonstrated the feasibility of directing evolution in natural Saccharomyces pastorianus hybrid stains in order to create variant strains with improved functional properties [6]. Such investigations have focused on adaptation to very high-gravity brewing conditions [7,8,9], associated stresses (such as osmotic stress and ethanol toxicity) [10,11], or the modification of the production of flavour compounds [12]. ALE has been utilised to enhance the fermentation rate of S. cerevisiae with decreased formation of acetate and greater production of aroma compounds [13,14]

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