The effect of microdosimetric 12C6+ heavy ion irradiation and Mg2+ on canthaxanthin production in a novel strain of Dietzia natronolimnaea

Abstract

Background: Dietzia natronolimnaea is one of the most important bacterial bioresources for high efficiency canthaxanthin production. It produces the robust and stable pigment canthaxanthin, which is of special interest for the development of integrated biorefineries. Mutagenesis employing 12C6+ irradiation is a novel technique commonly used to improve microorganism productivity. This study presents a promising route to obtaining the highest feasible levels of biomass dry weight (BDW), and total canthaxanthin by using a microdosimetric model of 12C6+ irradiation mutation in combination with the optimization of nutrient medium components.

Results: This work characterized the rate of both lethal and non-lethal dose mutations for 12C6+ irradiation and the microdosimetric kinetic model using the model organism, D. natronolimnaea svgcc1.2736. Irradiation with 12C6+ ions resulted in enhanced production of canthaxanthin, and is therefore an effective method for strain improvement of D. natronolimnaea svgcc1.2736. Based on these results an optimal dose of 0.5-4.5 Gy, Linear energy transfer (LET) of 80 keV μm-1and energy of 60 MeV u-1 for 12C6+ irradiation are ideal for optimum and specific production of canthaxanthin in the bacterium. Second-order empirical calculations displaying high R-squared (0.996) values between the responses and independent variables were derived from validation experiments using response surface methodology. The highest canthaxanthin yield (8.14 mg) was obtained with an optimized growth medium containing 21.5 g L-1 D-glucose, 23.5 g L-1 mannose and 25 ppm Mg2+ in 1 L with an irradiation dose of 4.5 Gy.

Conclusions: The microdosimetric 12C6+ irradiation model was an effective mutagenic technique for the strain improvement of D. natronolimnaea svgcc1.2736 specifically for enhanced canthaxanthin production. At the very least, random mutagenesis methods using 12C6+ions can be used as a first step in a combined approach with long-term continuous fermentation processes. Central composite design-response surface methodologies (CCD-RSM) were carried out to optimize the conditions for canthaxanthin yield. It was discovered D-glucose, Mg2+ and mannose have significant influence on canthaxanthin biosynthesis and growth of the mutant strain.

Figure 1

Survival of normal Dietzia natronolimnaea svgcc1.2736 strains after irradiation by ¹²C⁶⁺ion beams of different initial energies and LETs at dose levels of 0.5 to 5 Gy. (A) Surviving fraction of D. natronolimnaea svgcc1.2736 strains after irradiation with 60, 80, 100 and 120 keV/μm (LETs) and 30 MeV/u (energy) ¹²C⁶⁺-ions are compared. (B) Surviving fraction of D. natronolimnaea svgcc1.2736 strains after irradiation with 60, 80, 100 and 120 keV/μm (LETs) and 45 MeV/u (energy) ¹²C⁶⁺-ions are compared. (C) Surviving fraction of D. natronolimnaea svgcc1.2736 strains after irradiation with 60, 80, 100 and 120 keV/μm (LETs) and 60 MeV/u (energy) ¹²C⁶⁺-ions are compared. (D) Surviving fraction of D. natronolimnaea svgcc1.2736 strains after irradiation with 60, 80, 100 and 120 keV/μm (LETs) and 90 MeV/u (energy) 12C6+-ions are compared.

Figure 2

¹²C⁶⁺-ions of different parameters irradiation level and its effect on the growth rate of D. natronolimnaea smgcc1.2736 strains cells in %. (A-D) ¹²C⁶⁺-ions were accelerated up to 30 MeV/u, and their LETs were 60, 80, 100 and 120 keV/μm, with a dose rate of 0.5-1.5Gy. (E-H) ¹²C⁶⁺-ions were accelerated up to 45 MeV/u, and their LETs were 60, 80, 100 and 120 keV/μm, with a dose rate of 0.5-1.5Gy. (I-L) ¹²C⁶⁺-ions were accelerated up to 60 MeV/u, and their LETs were 60, 80, 100 and 120 keV/μm, with a dose rate of 0.5-1.5 Gy. (M-P) ¹²C⁶⁺-ions were accelerated up to 90 MeV/u, and their LETs were 60, 80, 100 and 120 keV/μm, with a dose rate of 0.5-1.5 Gy.

Figure 3

Influence of different irradiation dose (energy=45,60 MeV/u) on the D. natronolimnaeasvgcc1.2736 strains biomass dry weight and productivity. (A) LET for 60 keV/μm post-irradiation, 72 hours of cultivation illustrating the effect of biomass dry weight and specific productivity. (B) LET for 80 keV/μm post-irradiation, 72 hours of cultivation illustrating the effect of biomass dry weight and specific productivity. (C) LET for 100 keV/μm post-irradiation, 72 hours of cultivation illustrating the effect of biomass dry weight and specific productivity. (D) LET for 120 keV/μm post-irradiation, 72 hours of cultivation illustrating the effect of biomass dry weight and specific productivity.

Figure 4

Response surface curve (Left) and Contour plot (Right) of CX production by D. natronolimnaea svgcc1.2736 showing mutual interactions between showing mutual interactions between (A) D-glucose and mannose, (B) D-glucose and Mg²⁺, (C) ¹²C⁶⁺-ions irradiation dose and D-glucose. Other variables, except for those presented here, were maintained at zero.