Building up chemostats for experimental eco-evolutionary studies v2

Abstract

Chemostats and other flow through culture systems are a powerful tool for the study of microbial and plankton communities in experimental ecology and evolutionary studies. Commercially available chemostat systems allow the control of a large number of parameters (e.g., ph, pressure, CO 2 concentration) but are often expensive and offer a high level of control that is often not needed for many experimental studies. Non-commercial chemostats are more cost efficient, easily set up and flexible in volumes used. Different from semi-continuous culture systems, the flow through conditions of chemostats allow a constant inflow of resources from a reservoir (medium bottle) and outflow of unused nutrients, waste products and organisms, which are all collected in a waste bottle. Nutrient levels in the reservoir and the flow rate of the chemostat system (often referred to as dilution rate and presented as the fraction of the volume of the chemostat that is replaced per day) determines population growth rates and dynamics (for the theory behind chemostats see Smith & Waltman, 1995 and Weitz, 2015). Chemostats have been used in a number of studies and with different organisms and combinations of organisms. For example, Boer et al. (2010) studied how growth-limiting intracellular metabolites control yeast growth under diverse nutrient availability (Saccharomyces cerevisiae growing under five different nutrient supply of nitrogen: carbon: phosphorus). Becks et al. (2005) used chemostat systems to show how changes in the flow rate of a chemostat system influences the population dynamics of a three species microbial system. Frickel et al. (2016) used chemostats to investigate eco-evolutionary dynamics in a coevolving host-virus system (algae Chlorella variabilis and Chlorovirus strain PBCV-1) for 90 days. We present here an instruction for a cost efficient and flexible chemostat systems. These chemostats are composed of four main parts: a syringe unit, a glass bottle (i.e. the chemostat), a medium bottle and a waste bottle, all connected by tubing. A peristaltic pump and a low overpressure in the system allow the flow of medium from the reservoir to the chemostat bottle and of unused nutrients, waste products and organisms to the waste bottle. Chemostats are put on stirring plates to create a homogenosus environment within.

Evolution of exploitation and replication of giant viruses and virophages

“…We established the marine bacterivore Cafeteria burkhardae , the giant virus Cafeteria roenbergensis virus (CroV), and the virophage mavirus ( Fenchel and Patterson 1988 ; Fischer et al. 2010 ; Fischer and Suttle 2011 ; Schoenle 2020 ) in chemostats ( del Arco, Woltermann, and Becks 2020 ). Hereafter, we refer to CroV as ‘virus’ and to mavirus as ‘virophage’.…”

Virophage replication mode determines ecological and evolutionary changes in a host-virus-virophage system

“…We assembled host-virus-virophage communities in chemostat systems with 400 mL f/2 enriched artificial seawater medium (Guillard and Ryther 1962) supplemented with 0.025% (w/v) yeast extract with a continuous flow through of 120 mL medium per day (= 0.3 dilution rate per day) (del Arco et al 2020). Chemostats ran for 50 days in a culture room at 18 ± 0.5 ºC.…”

Evolution of virus and virophage facilitates persistence in a tripartite microbial system

“…Our experimental system consisted of the marine heterotrophic flagellate Cafeteria burkhardae strain E4-10P as host and the virus Cafeteria roenbergensis virus (CroV) and the virophage mavirus. The experiment was performed in chemostats (glass bottles with 400 mL f/2 enriched artificial seawater medium (50) supplemented with 0.025% (w/v) yeast extract and 0.3 g/mL of Chloramphenicol) with a continuous flow through of 120 ml medium per day (= 0.3 dilution rate per day) (23). Chemostats ran for 57 days.…”

“…Here, we use experimental evolution to investigate processes that could contribute to the long-term persistence of host-virus-virophage systems by testing whether and how exploitation of giant viruses and virophages evolve over time. We established the marine bacterivore Cafeteria burkhardae, the giant virus CroV and the virophage mavirus (19)(20)(21)(22) in chemostats (hereafter, we refer to CroV as 'virus' and to mavirus as 'virophage', (23)) and studied host population dynamics over 57 days either in the absence of virus and virophage, or with the virus, or with virus and virophage. To test for persistence of the three species and for evolutionary changes in the virus and virophage, we re-isolated virus and virophage from the chemostats after about ~280 host generations.…”