High-throughput cultivation and screening platform for unicellular phototrophs

Abstract

BackgroundHigh-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria.ResultsIn this work we describe and test high-throughput methods with the model organism Synechocystis sp. PCC6803. The required technical automation for these processes was achieved with a Tecan Freedom Evo 200 pipetting robot. The cultivation was performed in 2.2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. Each microtiter-well within the chamber functions as a separate cultivation vessel with reproducible conditions. The automated measurement of various parameters such as growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have already been established and can be monitored during cultivation. Measurement of growth parameters can be used as inputs for the system to allow for periodic automatic dilutions and therefore a semi-continuous cultivation of hundreds of cultures in parallel. The system also allows the automatic generation of mid and long term backups of cultures to repeat experiments or to retrieve strains of interest.ConclusionsThe presented platform allows for high-throughput cultivation and screening of Synechocystis sp. PCC6803. The platform should be usable for many phototrophic microorganisms as is, and be adaptable for even more. A variety of analyses are already established and the platform is easily expandable both in quality, i.e. with further parameters to screen for additional targets and in quantity, i.e. size or number of processed samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0239-x) contains supplementary material, which is available to authorized users.

Design and validation of a parallelized micro‐photobioreactor enabling phototrophic bioprocess development at elevated throughput

“…In terms of process monitoring and control, sophisticated concepts for automated at-line analytics and integrated screening workflows became available recently (Radzun et al, 2015;Tillich et al, 2014). Though, state-of-the-art non-invasive online analytics remain exclusively available in microbial microbioreactors (Kensy et al, 2009;Weuster-Botz et al, 2005).…”

“…Mostly, the homogeneity of light and process conditions and thereby cultivation reproducibility across the discrete wells of an MTP represents a crucial issue. There is either a restriction to use transparent plates allowing for interference by extraneous light and well‐to‐well cross‐talk (Radzun et al, ; Tillich et al, ) or suboptimal properties of the illumination unit (Chen et al, ). In this context, inhomogeneous illumination patterns were described to display problems.…”

A framework for accelerated phototrophic bioprocess development: integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design

“…Only recently a strong demand for high throughput micro-photobioreactors has been identified, based on which some prototype systems have been developed [ 19 – 27 ]. To take full advantage of phototrophic microscale cultivation, supporting methodologies and technologies, such as simplified strain maintenance [ 28 ], high throughput analytics [ 29 , 30 ], and automated processing [ 20 , 31 ], are needed.…”

The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002

“…In this paper we present a simple method for the distinction of the different phenotypes of ethanol producing and “revertant” cells via fluorescence microscopy. The approach is based on a previously developed fluorescence microscopic method [ 7 ] which has since also been adapted for high throughput application [ 8 ]. It allows a simple and quick viability analysis for cyanobacteria single cells or viability in a mix.…”