Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

Abstract

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

Culture characteristics of the atmospheric and room temperature plasma-mutated Spirulina platensis mutants in CO2 aeration culture system for biomass production

“…By the static flask culture, the growth rate (0.058 g L À1 $d À1 ) and Chl content (1.07 mg$g À1 ) of 3-A10 were lower than wild strain (0.084 g L À1 $d À1 , 3.53 mg$g À1 ), but its carbohydrate content (26.1%) was higher than the wild strain (18.6%), 3-B2 had the similar growth rate (0.079 g L À1 $d À1 ) and Chl content (3.51 mg$g À1 ) as the wild strain, but this mutant showed the highest carbohydrate content (33.1%, w/w) and productivity among the four strains; 4-B3 had the highest growth rate (0.093 g L À1 $d À1 ) and Chl content (4.52 mg$g À1 ), but its carbohydrate content (13.1%, w/w) was lower than that of the wild strain (16). These mutants showed high genetic stability by the subculture (16).…”

“…Typical S. platensis mutants of 3-A10, 3-B2 and 4-B3 strains were generated by the ARTP mutagenesis system combined with high throughput screening and maintained by the static flask culture reported previously by Fang et al (16). These S. platensis strains were cultivated in 250 ml flask with 120 ml fresh Zarrouk's medium (30) and 30 ml inoculum.…”

“…The optical density of S. platensis was determined at 560 nm (16,31) with UV spectrophotometer (UV757CRT, Shanghai, China), and converted to the dry cell weight (DCW) by using Eq. 1, where x and y mean DCW and OD 560 , respectively (16).…”

“…While the physical and chemical mutagenesis approaches used in S. platensis mutation have their own shortages due to the limited efficiency and safety. Recently, a new mutagenesis tool e atmospheric and room temperature plasmas (ARTP) developed by our group (15e18) has been successfully applied to create the mutant library of S. platensis (16). Moreover, ARTP mutagenesis system has been successfully applied in various microorganisms to improve diverse traits.…”

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis

“…Recently, a novel mutagenic breeding method, atmospheric and room temperature plasma (ARTP), has attracted general interest because its features include rapid mutation, highly diverse mutants, and simple and safe operation (Li et al 2012;Zhang et al 2014a, b). ARTP has been used for phenotype improvement (e.g., in productivity, toxicity tolerance, growth rate) in more than 40 kinds of microorganisms (Fang et al 2013;Hua et al 2010;Jiang et al 2014;Wang et al 2010;Zhang et al 2014a). However, there has been no quantitative evaluation or comparison of the DNA damage and consequent mutation rate caused by these different mutagenic sources.…”