Mesostats—A multiplexed, low-cost, do-it-yourself continuous culturing system for experimental evolution of mesocosms

Abstract

Microbial experimental evolution allows studying evolutionary dynamics in action and testing theory predictions in the lab. Experimental evolution in chemostats (i.e. continuous flow through cultures) has recently gained increased interest as it allows tighter control of selective pressures compared to static batch cultures, with a growing number of efforts to develop systems that are easier and cheaper to construct. This protocol describes the design and construction of a multiplexed chemostat array (dubbed “mesostats”) designed for cultivation of algae in 16 concurrent populations, specifically intended for studying adaptation to herbicides. We also present control data from several experiments run on the system to show replicability, data illustrating the effects of common issues like leaks, contamination and clumps, and outline possible modifications and adaptations of the system for future research.

Glyphosate resistance evolution to lethal and sublethal doses in chemostat populations of model organismChlamydomonas reinhardtii

“…Prior to the experiment, the algae had been kept in static stock cultures for two years (Ebert algal medium, 25°C, 24h light), with batches being transferred to fresh medium fortnightly, then in continuous flow culture for two months under the experiment control conditions detailed below. All C. reinhardtii populations were then cultured in continuous flow through chemostats (see [39] for detailed protocol) in Ebert algal medium at a dilution rate of 0.15/day with a shared multichannel peristaltic pump (Watson-Marlow 205S/CA16) controlling the flow for all culture chambers, maintaining a culture volume of 380 ml ± 5%. The populations were kept on a light box providing white light from below and surrounded on all sides with white light fluorescent bulbs at a light level of 75 μmol m -2 s -1 24 h a day.…”

Evidence for a trade-off between glyphosate resistance and anti-grazer defence in green algaChlamydomonas reinhardtii

“…16 Chlamydomonas reinhardtii strain Sager's CC-1690 wild-type 21 populations were cultured in a continuous flow-through chemostats (following Hansson et al, 2022) in sterile Ebert algal medium (Ebert, 2013) at a dilution rate of 0.15/24h with a shared multichannel peristaltic pump (Watson-Marlow 205S/CA16). Prior to the experiment, the algae had been kept in static stock 4 cultures for two years (Ebert algal medium, 25°C, 24h light), with batches being transferred to fresh medium fortnightly, then in continuous flow culture for two months under the experiment control conditions.…”