Microalgae are known to be a potential resource of high-value metabolites that can be used in the growing field of biotechnology. These metabolites constitute valuable compounds with a wide range of applications that strongly enhance a bio-based economy. Among these metabolites, astaxanthin is considered the most important secondary metabolite, having superior antioxidant properties. For commercial feasibility, microalgae with enhanced astaxanthin production need to be developed. In this study, the tropical green microalgae strain, Coelastrum sp., isolated from the environment in Malaysia, was incubated with methyl viologen, a reactive oxygen species (ROS) reagent that generates superoxide anion radicals (O2-) as an enhancer to improve the accumulation of astaxanthin. The effect of different concentrations of methyl viologen on astaxanthin accumulation was investigated. The results suggested that the supplementation of methyl viologen at low concentration (0.001 mM) was successfully used as a ROS reagent in facilitating and thereby increasing the production of astaxanthin in Coelastrum sp. at a rate 1.3 times higher than in the control.

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.

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.

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.