Gravity-Driven Adaptive Evolution of an Industrial Brewer's Yeast Strain towards a Snowflake Phenotype in a 3D-Printed Mini Tower Fermentor
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
... w 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. Fermentation 2017, 3, 4 2 of 12 Continuous culture provides many benefits over classical batch-style cultivation to perform experimental evolution  . Steady-state cultures allow for precise control of growth rate and environment, and cultures can be propagated for weeks or months in these controlled environments, which is important for the study of experimental evolution. Continuous mini bioreactors have been successfully used as multiplexed chemostat arrays for adaptive evolution experiments with yeast cells [15, 16] . The use of mini bioreactors has several advantages, such as reduced costs for media and labour, and the ability to perform a large number of fermentations in parallel    . Recently, three-dimensional (3D) printing has been used for medical applications , such as (1) medical models; (2) medical aids, orthoses, splints, and prostheses; (3) tools, instruments, and parts for medical devices; (4) inert implants; and (5) biomanufacturing     . Bioprocess applications include tissue engineering scaffolds and corresponding bioreactors  . 3D printing has recently been used to fabricate fullerene-type biocarriers for biofilm growth that can be used in bioreactors for wastewater treatment  . 3D printing technology shows great potential for the easy development of mini-scale bioreactors. Here, we report the use of 3D printing for the construction of a continuous mini tower fermentor. The concept of 3D printing-also referred to as additive manufacturing, rapid prototyping, or solid-freeform fabrication (SFF)-was developed by Charles Hull in the early 1980's  . 3D printing is used for rapid prototyping of 3D models originally generated by a computer aided design (CAD) program. The 3D model is sliced into 2D horizontal cross sections, which are printed in consecutive layers. There are several well-established methods of 3D-printing, such as stereolithography (SLA), fused deposition modelling, selective laser sintering, multi jet fusion, and selective laser melting. SLA became the first commercialised 3D-printing technique (invented by Charles Hull in 1983), and remains one of the most powerful and versatile of all SFF techniques  . SLA works by exposing a layer of photosensitive liquid resin to a UV-laser beam so that the resin hardens and becomes solid. Once the laser has swept a layer of resin in the desired pattern and it begins to harden, the model-building platform in the liquid tank of the printer steps down the thickness of a single layer, and the laser begins to form the next layer. SLA is capable of printing at high resolutions (up to 25 µm) and relatively high production rates (1.5 cm/h). In this contribution, we designed a continuous mini bioreactor that is suitable to perform ALE of a weak flocculating industrial brewers' strain towards a strain with a more extended aggregation (flocculation) phenotype. Yeast strains that aggregate or flocculate are of particular interest to the brewer, since it simplifies yeast removal at the end of the primary fermentation  . Since many industrial brewing strains still show no adequate flocculation, we applied ALE as a non-genetic engineering method. The design of the fermentor was based on a continuous mini tower fermentor; i.e., the continuous A.P.V. tower fermentor that was used in the 1960s to produce beer on an industrial scale in a British brewery [30, 31] . In this type of fermentor, gravity is the selective pressure to enhance the aggregation phenotype during evolution. 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 ("snowflake") phenotype by performing ALE. Materials and Methods Yeast Strains and Media An industrial S. cerevisiae brewer's strain was used; i.e., CMBSVM22 from the yeast collection of prof. em. Freddy Delvaux (Centre for Malting and Brewing Science (CMBS), Katholieke Universiteit Leuven, Leuven Belgium; Biercentrum Delvaux, Beekstraat 20, 3040 Neerijse, Belgium) . The haploid S. cerevisiae BY4742 strain  was used for the biocompatibility assay, and the flocculent BY4742 [FLO1] strain  for the preliminary experiments during experimental design optimisation. These strains were precultured in YPD (Yeast extract-Peptone-Dextrose) medium (1% m/v yeast extract, 2% m/v meat peptone, 4% m/v D-glucose) overnight at 30 • C. Growth medium (100 g/L D-glucose, Fermentation 2017, 3, 4 9 of 12 Conclusions A continuous mini tower fermentor was designed and constructed using a 3D printer. The printed fermentor was biocompatible for yeast cultivation and could be sterilized by autoclaving. The tower fermentor was suitable to perform experimental evolution experiments where gravity acts as the selective pressure, and allowed yeast cells to evolve from planktonic single cells towards cell aggregates. After 14 days of continuous cultivation of a non-flocculating industrial brewer's yeast strain in the fermentor, yeast flocs with a "snowflake" morphology were observed. Yeast floc characterisation showed that the cell-cell interactions in the cell aggregates 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.