Microalgae have long been used for the commercial production of natural colorants such as carotenoids and chlorophyll. Due to the rising demand for carotenoids and other natural products from microalgae, strategies to increase production efficiency are urgently needed. The production of microalgal biorefineries has been limited to countries with moderate climates. For countries with cooler climates and less daylight, methodologies for the efficient production of microalgal biorefineries need to be investigated. Algal strains that can be safely consumed as whole cells are also attractive alternatives for developing as carotenoid supplements, which can also contain other compounds with health benefits. Using such strains helps to eliminate the need for hazardous solvents for extraction and several other complicated steps. In this study, the mesophilic green alga Chlamydomonas reinhardtii was employed to study the effects of cold stress on cell physiology and the production of pigments and storage compounds. The results showed that temperatures between 10 and 20 °C induced carotenoid and chlorophyll accumulation in the wild-type strain of C. reinhardtii. Interestingly, the increased level of carotenoids suggested that they might play a crucial role in cold stress acclimation. A temperature of 15 °C resulted in the highest carotenoid and chlorophyll productivity. At this temperature, carotenoid and chlorophyll productivity was 2 times and 1.3 times higher than at 25 °C, respectively. Subjecting a mutant defective in lutein and zeaxanthin accumulation to cold stress revealed that these two carotenoids are not essential for cold stress survival. Therefore, cold temperature could be used as a strategy to induce and increase the productivity of pigments in C. reinhardtii.

Sterols and squalene are essential biomolecules required for the homeostasis of eukaryotic membrane permeability and fluidity. Both compounds have beneficial effects on human health. As the current sources of sterols and squalene are plant and shark oils, microalgae are suggested as more sustainable sources. Nonetheless, the high costs of production and processing still hinder the commercialization of algal cultivation. Strain improvement for higher product yield and tolerance to harsh environments is an attractive way to reduce costs. Being an intermediate in sterol synthesis, squalene is converted to squalene epoxide by squalene epoxidase. This step is inhibited by terbinafine, a commonly used antifungal drug. In yeasts, some terbinafine-resistant strains overproduced sterols, but similar microalgae strains have not been reported. Mutants that exhibit greater tolerance to terbinafine might accumulate increased sterols and squalene content, along with the ability to tolerate the drug and other stresses, which are beneficial for outdoor cultivation. To explore this possibility, terbinafine-resistant mutants were isolated in the model green microalga Chlamydomonas reinhardtii using UV mutagenesis. Three mutants were identified and all of them exhibited approximately 50 percent overproduction of sterols. Under terbinafine treatment, one of the mutants also accumulated around 50 percent higher levels of squalene. The higher accumulation of pigments and triacylglycerol were also observed. Along with resistance to terbinafine, this mutant also exhibited higher resistance to oxidative stress. Altogether, resistance to terbinafine can be used to screen for strains with increased levels of sterols or squalene in green microalgae without growth compromise.

Biodiesel and natural products derived from microalgae require a smaller land area and have higher production rates compared to plants and animals and has recently attracted considerable interest. However, biodiesel production from microalgal triacylglycerol is still far from commercial realization due to its high production cost. One way to overcome this obstacle is to improve the triacylglycerol accumulation and couple its production with other high-value compounds. Of particular interest is the sterol biosynthetic pathway with squalene as an intermediate due to its close relationship with triacylglycerol and carotenoid biosynthetic pathways. Besides, both squalene and carotenoids are isoprenoid lipids that have health benefits. Perturbation of one pathway has been suggested to affect other pathways. Three terbinafine-sensitive mutants of the green microalga Chlamydomonas reinhardtii were isolated using terbinafine, a drug that inhibits squalene epoxidase, leading to squalene accumulation. One of the mutants, tfs2, accumulated twice the amount of wild-type triacylglycerol. As well as squalene accumulation, the presence of terbinafine further increased the triacylglycerol content. The level of prenyl lipid carotenoid and chlorophyll was also more significant than that of the wild type. Growth and photosynthesis were not compromised in this mutant. This is the first study that has demonstrated a mutant screening method to improve the co-production of TAG and isoprenoid lipids in a green microalgae.

Photosynthetic organisms, such as higher plants and algae, require light to survive. However, an excessive amount of light can be harmful due to the production of reactive oxygen species (ROS), which cause cell damage and, if it is not effectively regulated, cell death. The study of plants’ responses to light can aid in the development of methods to improve plants’ growth and productivity. Due to the multicellular nature of plants, there may be variations in the results based on plant age and tissue type. Chlamydomonas reinhardtii, a unicellular green alga, has also been used as a model organism to study photosynthesis and photoprotection. Nonetheless, the majority of the research has been conducted with strains that have been consistently utilized in laboratories and originated from the same source. Despite the availability of many field isolates of this species, very few studies have compared the light responses of field isolates. This study examined the responses of two field isolates of Chlamydomonas to high light stress. The light-tolerant strain, CC-4414, managed reactive oxygen species (ROS) slightly better than the sensitive strain, CC-2344, did. The proteomic data of cells subjected to high light revealed cellular modifications of the light-tolerant strain toward membrane proteins. The morphology of cells under light stress revealed that this strain utilized the formation of palmelloid structures and cell aggregation to shield cells from excessive light. As indicated by proteome data, morphological modifications occur simultaneously with the increase in protein degradation and autophagy. By protecting cells from stress, cells are able to continue to upregulate ROS management mechanisms and prevent cell death. This is the first report of palmelloid formation in Chlamydomonas under high light stress.

Numerous species of microalgae have been utilized for pigment production. More and more species are gaining popularity due to their ability to accumulate pigments with varying chemical compositions and the fact that some have distinctive byproducts that can be co-produced. Despite the fact that many of the species have unique by-products and traits, they are not being used economically due to high production costs. Utilizing agricultural and industrial wastewater for algae cultivation is one way to lower manufacturing costs. Herbicide-contaminated wastewater can result from agricultural contamination. Norflurazon is a popular pesticide frequently used for weed control. The presence of norflurazon in water renders that water unusable and requires proper treatment. Nanoparticles of ZnO (ZnO NPs), on the other hand, are utilized in a variety of industrial productions of numerous household goods. Water contaminated with ZnO NPs can present potential risks to human health and the environment. In this study, two field isolates of the green microalga Chlamydomonas reinhardtii, a widely used model organism, were examined for their reaction to these two compounds in order to assess the responses of different natural strains to environmental stresses. Norflurazon at 10 µM had a higher inhibitory effect on growth and pigment production than ZnO NPs at 200 mg L⁻¹. Although both norflurazon and ZnO NPs inhibit cell growth and pigmentation, they do so through distinct processes. Norflurazon induces oxidative stress in cells, resulting in photosystem damage. ZnO nanoparticles, on the other hand, did not cause photosystem damage but rather mechanical cell damage and disintegration. In addition, the physiological responses of the two Chlamydomonas strains were distinct, supporting the utilization of natural algal strains for specific types of environmental pollutants.

Norflurazon is a widely utilized pesticide in agriculture for weed management. The mechanism of action involves the inhibition of an initial step in carotenoid synthesis. This inhibition results in the instability of the photosynthetic machinery and subsequent cell bleaching. Microalgae have attracted significant interest for the production of valuable products. Nonetheless, the mass cultivation of microalgae continues to encounter many challenges that result in high production costs. A potential issue in photobioreactor and open pond cultivation is contamination by other microalgae, which can destroy the mass culture entirely. Strains exhibiting greater resistance to specific chemicals may be beneficial in reducing contamination from other algae. Furthermore, integrating microalgal production with phycoremediation constitutes a sustainable approach to the circular economy. Many norflurazon-resistant microalgae strains have been developed, including the model unicellular green microalga Chlamydomonas reinhardtii. In previous studies, mutant and transgenic strains resistant to high concentrations of norflurazon have been generated to study herbicide tolerance in Chlamydomonas reinhardtii. Nonetheless, the application of genetically engineered organisms should remain cautious. Moreover, mutant strains generated through conventional methods were created using very high chemical concentrations. The effects of introducing such strains on the composition of organisms in the environment remain a concern. This study investigated the feasibility of utilizing natural isolates of this alga for mass production in the presence of norflurazon. Twenty isolates of this alga were evaluated for tolerance to norflurazon. The two most tolerant isolates demonstrated the ability to withstand 5–10 µM of norflurazon, a concentration previously employed to select mutants and transformants resistant to norflurazon. Physiological and proteomic data revealed an enhancement of photosynthesis and photoprotection processes as the primary mechanism for norflurazon tolerance in one isolate, whereas another isolate demonstrated a reduction in protein synthesis, photosynthesis, and cell motility.