Robust, small-scale cultivation platform for Streptomyces coelicolor

Abstract

Background: For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes.

Results: To address these issues, we devised a robust, small-scale cultivation platform in the form of MTPs (24-square deepwell) for the filamentous bacterium Streptomyces coelicolor and compared its performance to that of shake flasks and bench-scale reactors. We observed that re-designing of medium and inoculum preparation recipes resulted in improved reproducibility. Process turnaround time was significantly reduced due to the reduction in number of unit operations from inoculum to cultivation. The incorporation of glass beads (ø 3 mm) in MTPs not only improved the process performance in terms of improved oxygen transfer improving secondary metabolite production, but also helped to transform morphology from pellet to disperse, resulting in enhanced reproducibility. Addition of MOPS into the medium resulted in pH maintenance above 6.50, a crucial parameter towards reproducibility. Moreover, the entire trajectory of the process was analyzed for compatibility with bench-scale reactors. The MTP cultivations were found to behave similar to bench-scale in terms of growth rate, productivity and substrate uptake rate and so was the onset of antibiotic synthesis. Shake flask cultivations however, showed discrepancy with respect to morphology and had considerably reduced volumetric production rates of antibiotics.

Conclusion: We observed good agreement of the physiological data obtained in the developed MTP platform with bench-scale. Hence, the described MTP-based screening platform has a high potential for investigation of secondary metabolite biosynthesis in Streptomycetes and other filamentous bacteria and the use may significantly reduce the workload and costs.

Figure 1

Preparation of frozen mycelia. Frozen mycelium was prepared from a spore plate of S. coelicolor incubated for 8 days. Boxes with grey filling represent quality control steps.

Figure 2

Effect of bead size on S. coelicolor morphology. Each MTP containing beads of one of the following sizes (no beads, 0.75, 2, 3 and 4 mm) was inoculated with S. coelicolor. About 1 ml sample (taken at 48 hrs) from each MTP was loaded into the mixing chamber of Mastersizer 2000 to determine the size distribution. Data shown are mean values from three wells and error bars represent standard deviation.

Figure 3

Morphology of S. coelicolor across scales. a) Particle size distributions at two different time points during growth in MTP. Samples were taken at 72 and 96 hrs from three wells and mean values for each time point is reported. b) Reproducibility across wells in MTPs. Samples was taken from three wells at 72 hrs to estimate mean values. (c) Particle size distribution in bioreactor cultivations. Three samples were taken at each time point (37, 53 and 74 hrs). Mean values are presented in the figure.

Figure 4

Effect of MOPS buffer on a) Actinorhodin titer b) Specific growth rate. Five different MOPS concentrations (50, 100, 130, 160 and 190 mM) were investigated. The concentration of actinorhodin described in the figure is from 144 hrs. Samples from 0 to 48 hrs were used to estimate specific growth rate. Data presented are mean values of three samples. Please note that data from cultivations with 50 mM MOPS was not included in the figure as pH decreased to 5.80 early during cultivation, severely affecting growth and production.

Figure 5

Batch cultivation profile in the optimized MTP. S. coelicolor was cultivated in MTPs containing 3 ml defined medium with 100 mM MOPS and 3 mm glass beads. Typical time course profiles for the parameters biomass in the form of OD_{450 nm} (♦) and DCW (■) g/L, glucose (●) g/L, UDP (Δ) mg/L and ACT (▲) mg/L and pH (--) are shown. OD, DCW, ACT and pH trends are represented on primary axis while, glucose and UDP trends are depicted on secondary axis.

Figure 6

Batch cultivation profile in the reactor. S. coelicolor was cultivated in a reactor with 1 L defined medium. Typical time course profiles for the parameters biomass in the form of OD_{450 nm} (♦) and DCW (■) g/L, glucose (●) g/L, UDP (Δ) mg/L, ACT (▲) mg/L and pH (--) are shown. OD, DCW, ACT and pH trends are represented on primary axis while, glucose and UDP trends are depicted on secondary axis.