Assessment of Domestic Wastewaters as Potential Growth Media for Chlorella vulgaris and Haematococcus pluvialis

Domestic wastewater contains chemical compounds that can be used as nutrients for microalgae. Removing these chemical compounds from wastewater by microalgae might help in reducing the operation cost of wastewater management while minimizing the cultivation cost for large-scale microalgae metabolite production. In this study, domestic wastewater collected from Indah Water Konsortium (IWK), Kuala Lumpur, Malaysia, was assessed as growth media for two types of microalgae, namely Chlorella vulgaris and Haematococcus pluvialis. The biomass growth and nutrient removal efficiency of total nitrogen (TN), total phosphorus (TP), and total ammonia (TAN) in different concentrations of diluted wastewater were measured. The results showed that biomass concentration (0.227 g/L), biomass productivity (0.029 g/L/day), and specific growth rate (0,284 d-1) yielded by C. vulgaris in 14 days of 80% wastewater were comparable to those microalgae grew in standard Bold’s Basal medium (BBM). Besides, C. vulgaris grew in 50% wastewater to remove TN, TP, and TAN with the highest removal efficiency (>88%). For H. pluvialis, the biomass concentration in all wastewater concentrations was lower than BBM. The removal efficiencies of TN and TP were lower than 55%, but more than 80% for removal efficiency of TAN in 50% and 80% wastewater. Hence, C. vulgaris has better growth performance and nutrient removal efficiency than H. pluvialis. These findings indicated that IWK domestic wastewater could be used as growth media for microalgae, especially C. vulgaris.

Producción de astaxantina bajo factores de estrés utilizando un biorreactor a escala de laboratorio de 5 L

Introducción. Haematococcus pluvialis es una microalga que produce astaxantina, un betacaroteno y antioxidante muy usado en la industria. Para obtener una mayor producción de astaxantina se planteó como Objetivo utilizar diferentes factores de estrés, en un biorreactor a escala de laboratorio de 5 litros. Metodología. Se cultivó la microalga en el medio RM, pH 6,8, temperatura 20±2ºC, aire filtrado, iluminación con lámparas blancas 20h luz/4h oscuridad, irradianza 70 μE m−2s−1, diferentes concentraciones de acetato de sodio y cloruro de sodio. Se determinó crecimiento celular, cambios morfológicos y cuantificación de astaxantina y clorofila por espectrofotometría. Se realizó un análisis estadístico utilizando ANOVA (95%). Resultados. Utilizando 0,299 mg/L de acetato de sodio se obtuvo un crecimiento celular de 2,0 x 104 Cel/mL y una concentración de astaxantina de 2,530 µg/mL, mientras que con 1,6 mg/L de acetato de sodio el crecimiento celular fue de 3,5 x 104 Cel/mL y una concentración de astaxantina de 1,9 µg/ml. El tratamiento al cual se le adicionó 1,6 g/L de acetato de sodio y 6,4 g/L de cloruro de sodio presentó la mayor producción astaxantina 7,3 µg/ml. El tratamiento con acetato de sodio 0,320 g/L + cloruro de sodio 1,28 g/L presentó el mayor crecimiento celular con 1,64x105 células/ml. Conclusión. Esta investigación destaca la importancia de cultivar inicialmente la microalga utilizando el biorreactor Tecferm de 5 litros y después de su fase exponencial someterla a factores de estrés con acetato de sodio y cloruro de sodio lográndose así la mayor producción de astaxantina 7,325 µg/ml.

Transcriptomic and Proteomic Characterizations of the Molecular Response to Blue Light and Salicylic Acid in Haematococcus pluvialis

Haematococcus pluvialis accumulates a large amount of astaxanthin under various stresses, e.g., blue light and salicylic acid (SA). However, the metabolic response of H. pluvialis to blue light and SA is still unclear. We investigate the effects of blue light and SA on the metabolic response in H. pluvialis using both transcriptomic and proteomic sequencing analyses. The largest numbers of differentially expressed proteins (DEPs; 324) and differentially expressed genes (DEGs; 13,555) were identified on day 2 and day 7 of the treatment with blue light irradiation (150 μmol photons m−2s−1), respectively. With the addition of SA (2.5 mg/L), a total of 63 DEPs and 11,638 DEGs were revealed on day 2 and day 7, respectively. We further analyzed the molecular response in five metabolic pathways related to astaxanthin synthesis, including the astaxanthin synthesis pathway, the fatty acid synthesis pathway, the heme synthesis pathway, the reactive oxygen species (ROS) clearance pathway, and the cell wall biosynthesis pathway. Results show that blue light causes a significant down-regulation of the expression of key genes involved in astaxanthin synthesis and significantly increases the expression of heme oxygenase, which shows decreased expression by the treatment with SA. Our study provides novel insights into the production of astaxanthin by H. pluvialis treated with blue light and SA.

Fenton Reaction Facilitates The Fungal Infection On The Unicellular Alga Haematococcus Pluvialis and Discovery of a Biosafe and Environment-Friendly Chemical That Blocks The Infection

Background The green microalga Haematococcus pluvialis is used as cell factories for producing astaxanthin, the high-value natural compound with multiple biological functions. However, H. pluvialis is prone to the infection by a parasitic fungus Paraphysoderma sedebokerense, which is the most devastating threat to the mass culture of H. pluvialis all over the world. Little is known for the mechanisms underlying the infection process, though it is of great essential for developing effective measures to mitigate the pathogen threatening for the natural astaxanthin industry. Results We observed that there were heat-stable substances with small molecular weight produced during the infection process, which significantly stimulated the parasitism process and enhanced the susceptibility of H. pluvialis cells to the pathogen. Systematic approaches including multi-omics, biochemical and imaging analysis were deployed to uncover the identity of the metabolites and the underlying mechanisms. Two metabolites, 3-hydroxyanthranilic acid and hordenine were identified and proved to stimulate the infection via driving fenton reaction mediated oxidative stress to H. pluvialis. The reaction generated hydroxyl radicals to disrupt the subcellular components of the algal cells and to make the algal cells more susceptible to the infection. Based on these findings, a biosafe and environment-friendly antioxidant butylated hydroxyanisole was selected to inhibit the fungal infection, which completely abolished the infection at 12 ppm. Conclusions This study provide for the first time, a framework to dissect the functions of secondary metabolites in the interaction between the unicellular algal H. pluvialis and its fungi parasite, indicating that oxidative degradation is a strategy used for the fungal infest. Eliminating the oxidative burst through adding antioxidant butylated hydroxyanisole could be an effective measure to reduce parasitic infection in H. pluvialis mass culture.

Characterization of a Haematococcus pluvialis Diacylglycerol Acyltransferase 1 and Its Potential in Unsaturated Fatty Acid-Rich Triacylglycerol Production

The unicellular green alga Haematococcus pluvialis has been recognized as an industry strain to produce simultaneously esterified astaxanthin (EAST) and triacylglycerol (TAG) under stress induction. It is necessary to identify the key enzymes involving in synergistic accumulation of EAST and TAG in H. pluvialis. In this study, a novel diacylglycerol acyltransferase 1 was systematically characterized by in vivo and in silico assays. The upregulated expression of HpDGAT1 gene was positively associated with the significant increase of TAG and EAST contents under stress conditions. Functional complementation by overexpressing HpDGAT1 in a TAG-deficient yeast strain H1246 revealed that HpDGAT1 could restore TAG biosynthesis and exhibited a high substrate preference for monounsaturated fatty acyl-CoAs (MUFAs) and polyunsaturated fatty acyl-CoAs (PUFAs). Notably, heterogeneous expression of HpDGAT1 in Chlamydomonas reinhardtii and Arabidopsis thaliana resulted in a significant enhancement of total oils and concurrently a high accumulation of MUFAs- and PUFAs-rich TAGs. Furthermore, molecular docking analysis indicated that HpDGAT1 contained AST-binding sites. These findings evidence a possible dual-function role for HpDGAT1 involving in TAG and EAST synthesis, demonstrating that it is a potential target gene to enrich AST accumulation in this alga and to design oil production in both commercial algae and oil crops.