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Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

A newly isolated green microalga, Coelastrum sp. has the capability to produce and accumulate astaxanthin under various stress conditions. At present, a mutant G1-C1 of Coelastrum sp. obtained through chemical mutagenesis using ethyl methane sulfonate displayed an improvement in astaxanthin accumulation, which was 2-fold higher than that of the wild-type. However, lack of genomic information limits the understanding of the molecular mechanism that leads to a high level of astaxanthin in the mutant G1-C1. In this study, transcriptome sequencing was performed to compare the transcriptome of astaxanthin hyper-producing mutant G1-C1 and wild-type of Coelastrum sp. with respect to astaxanthin biosynthesis. This is to clarify why the mutant produced higher astaxanthin yield compared to the wild-type strain. Based on the transcriptomic analysis, the differentially expressed genes involved in astaxanthin biosynthesis were significantly upregulated in the mutant G1-C1 of Coelastrum sp. Genes coding phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase involved in β-carotene biosynthesis in the mutant cells were upregulated by 10-, 9.2-, 8.4-, and 8.7-fold, respectively. Genes coding beta-carotene ketolase and beta-carotene 3-hydroxylase involved in converting β-carotene into astaxanthin were upregulated by 7.8- and 8.0-fold, respectively. In contrast, the lycopene ε-cyclase gene was downregulated by 9.7-fold in mutant G1-C1. Together, these results contribute to higher astaxanthin accumulation in mutant G1-C1. Overall, the data in this study provided molecular insight for a better understanding of the differences in astaxanthin biosynthesis between the wild-type and mutant G1-C1 strains.

Highlights • A newly isolated Coelastrum sp. improved the yield of astaxanthin by chemical mutagenesis. • Mutagenesis using chemical mutagen of EMS attempted to increase the microalgae biomass and carotenoid production in Coelastrum sp. • High-throughput screening method using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp. • The selected mutant using glufosinate exhibited an increase of astaxanthin content with ∼2-fold higher compared to the wild type., Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

Astaxanthin is one of the most important secondary metabolites with superior antioxidant property and is widely applied in nutraceuticals and pharmaceuticals industry. Recently, the production of astaxanthin from natural source has been mainly focused on the green microalgae, Haematococcus pluvialis, as it can accumulate large amounts of astaxanthin and its esters. However, the slow growth with low biomass yield and easy contamination by other fast-growing organisms are the main problems faced by H. pluvialis. In this study, the preliminary study on growth characteristic and amount of astaxanthin by tropical microalgae strains isolated from environment in Malaysia was reported. Two newly isolated microalgae including Acutodesmus obliquus and Coelastrum sp. were compared under various stress inductive conditions to identify the microalgae culture that can accumulate high amount of astaxanthin. The objective of this study is to compare and characterise astaxanthin accumulation using different species of green microalgae as mentioned above under different stress conditions. Findings of the preliminary studied indicated that the exposure of microalgae culture to high light intensity and nitrogen starvation in mixotrophic culture is a potential inducer of high amount of astaxanthin production. Among the two microalgae studied, Coelastrum sp., exhibited the highest tolerance to stress conditions. Outcomes of this work have shown that, astaxanthin produced in Coelastrum sp. is the most comparable to H. pluvialis and can be the potential alternative to current astaxanthin production.

The discharge of highly coloured azo dyes effluent has caused serious environmental damages. In this study, bacteria isolated from Antarctica seawater were screened for their ability to decolourise azo dye Reactive Black 5 (RB5). The selected bacterium was further investigated to study its ability to decolourise RB5. The best bacteria from Antarctica seawater that had the ability to decolourise RB5 was identified using 16S rDNA sequence analysis and revealed that the bacteria 15C shared 99% homology to Klebsiella pneumoniae. Selection of the most effective bacteria was followed by its acclimatisation to decolourise higher concentrations of RB5 by growing it in successively higher concentrations of RB5. Following that, optimization of RB5 decolourisation by the selected bacteria was performed using one factor at time (OFAT) including concentration of dye, pH and temperature. The results obtained indicated the optimal condition for decolourisation of RB5 using this bacterium was at pH 10 and 37°C in 70 mg/L RB5 with 98% decolourisation within 24 h under facultative anaerobic treatment. Besides that, 70% of COD removal was achieved after 96 h of sequential anaerobic and aerobic treatment of RB5. In addition, FTIR and HPLC were used to analyze the metabolite of RB5 decolourisation. Products of RB5 decolourisation was confirmed by the presence of sulphanilic acid in HPLC analyses and the changes observed in the functional groups of the FTIR spectrum suggesting the possibility of RB5 degradation. Ammonia test and carbon dioxide test showed higher concentration of ammonia and carbon dioxide that indicates the mineralisation of product after treatment.