Comprehensive Analysis of Biomass from Chlorella sorokiniana Cultivated with Industrial Flue Gas as the Carbon Source.

Chlorella sorokiniana, isolated from a pond adjacent to a cement plant, was cultured using flue gas collected directly from kiln emissions using 20 L and 25000 L photobioreactors. Lipids, proteins, and polysaccharides were analyzed to understand their overall composition for potential applications. The lipid content ranged from 17.97% to 21.54% of the dry biomass, with carotenoid concentrations between 8.4 and 9.2 mg/g. Lutein accounted for 55% of the total carotenoids. LC/MS analysis led to the identification of 71 intact triacylglycerols, 8 lysophosphatidylcholines, 10 phosphatidylcholines, 9 monogalactosyldiacylglycerols, 12 digalactosyldiacylglycerols, and 1 sulfoquinovosyl diacylglycerol. Palmitic acid, oleic acid, linoleic acid, and α-linolenic acid were the main fatty acids. Polyunsaturated fatty acid covers ≥ 56% of total fatty acids. Protein isolates and polysaccharides were also extracted. Protein purity was determined to be ≥75% by amino acid analysis, with all essential amino acids present. Monomer analysis of polysaccharides suggested that they are composed of mainly D-(+)-mannose, D-(+)-galactose, and D-(+)-glucose. The results demonstrate that there is no adverse effect on the metabolite profile of C. sorokiniana biomass cultured using flue gas as the primary carbon source, revealing the possibility of utilizing such algal biomass in industrial applications such as animal feed, sources of cosmeceuticals, and as biofuel.

Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana.

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1-5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

The Impact of Calcium Depletion on Proliferation of Chlorella sorokiniana Strain DSCG150.

This study analyzed the effects of Ca2+ metal ions among culture medium components on the Chlorella sorokiniana strain DSCG150 strain cell growth. The C. sorokiniana strain DSCG150 grew based on a multiple fission cell cycle and growth became stagnant in the absence of metal ions in the medium, particularly Ca2+. Flow cytometry and confocal microscopic image analysis results showed that in the absence of Ca2+, cell growth became stagnant as the cells accumulated into four autospores and could not transform into daughter cells. Genetic analysis showed that the absence of Ca2+ caused upregulation of calmodulin (calA) and cell division control protein 2 (CDC2_1) genes, and downregulation of origin of replication complex subunit 6 (ORC6) and dual specificity protein phosphatase CDC14A (CDC14A) genes. Analysis of gene expression patterns by qRT-PCR showed that the absence of Ca2+ did not affect cell cycle progression up to 4n autospore, but it inhibited Chlorella cell fission (liberation of autospores). The addition of Ca2+ to cells cultivated in the absence of Ca2+ resulted in an increase in n cell population, leading to the resumption of C. sorokiniana growth. These findings suggest that Ca2+ plays a crucial role in the fission process in Chlorella.

High biomass yields of Chlorella protinosa with efficient nitrogen removal from secondary effluent in a membrane photobioreactor.

Further treatment of secondary effluents before their discharge into the receiving water bodies could alleviate water eutrophication. In this study, the Chlorella proteinosa was cultured in a membrane photobioreactor to further remove nitrogen from the secondary effluents. The effect of hydraulic retention time (HRT) on microalgae biomass yields and nutrient removal was studied. The results showed that soluble algal products concentration reduced in the suspension at low HRT, thereby alleviating microalgal growth inhibition. In addition, the lower HRT reduced the nitrogen limitation for Chlorella proteinosa's growth through the phase-out of nitrogen-related functional bacteria. As a result, the productivity for Chlorella proteinosa increased from 6.12 mg/L/day at an HRT of 24 hr to 20.18 mg/L/day at an HRT of 8 hr. The highest removal rates of 19.7 mg/L/day, 23.8 mg/L/day, and 105.4 mg/L/day were achieved at an HRT of 8 hr for total nitrogen (TN), ammonia, and chemical oxygen demand (COD), respectively. However, in terms of removal rate, TN and COD were the largest when HRT is 24 hr, which were 74.5% and 82.6% respectively. The maximum removal rate of ammonia nitrogen was 99.2% when HRT was 8 hr.

Microalgae cultivation trials in a membrane bioreactor operated in heterotrophic, mixotrophic, and phototrophic modes using ammonium-rich wastewater: The study of fouling.

In this work, microalgae cultivation trials were carried out in a membrane bioreactor to investigate fouling when the cultures of Chlorellavulgaris were grown under mixotrophic, heterotrophic, and phototrophic cultivation regimes. The Chlorella cultures were cultivated in wastewater as a source of nutrients that contained a high concentration of ammonium. In mixotrophic cultivation trials, the results showed that the elevated contents of carbohydrates in the soluble microbial product and proteins in extracellular polymeric substances probably initiated membrane fouling. In this case, the highest protein content was also found in extracellular polymeric substances due to the high nitrogen removal rate. Consequently, transmembrane pressure significantly increased compared to the phototrophic and heterotrophic regimes. The data indicated that cake resistance was the main cause of fouling in all cultivations. Higher protein content in the cake layer made the membrane surface more hydrophobic, while carbohydrates had the opposite effect. Compared to a mixotrophic culture, a phototrophic culture had a larger cell size and higher hydrophobicity, leading to less membrane fouling. Based on our previous data, the highest ammonia removal rate was reached in the mixotrophic cultures; nevertheless, membrane fouling appeared to be the fundamental problem.

Performance evaluation of typical flocculants for efficient harvesting of Chlorella sorokiniana under different carbon application modes.

In this study, the growth characteristics of microalgae cultured with different carbon sources were analyzed, and the flocculation characteristics under the influence of carbon sources were evaluated using three typical flocculants. The results showed that the organic carbon sources could significantly increase the content of extracellular proteins in microalgae. Specifically, the extracellular protein concentrations of microalgae cultured with pure BG-11, ethanol, sodium acetate and glucose were 18.2 29.2, 97.3, and 34.7 mg/g, respectively. During the flocculation process, microalgae cultured with sodium acetate exhibited a weak response to the flocculant because of excessive extracellular proteins inhibited flocculation. In addition, the flocculation efficiency was also less than 50.0% cultured with sodium acetate in all pH test ranges when alum and chitosan were used as flocculants. It could be inferred that the flocculant initially happened to charge neutralization with the negatively charged proteins in the solution and then bridged the charges with the microalgae. These findings provide insights into the effects of different carbon sources on microalgal flocculation, promising organic integration of microalgae wastewater treatment and harvesting.

Effects of sulfamonomethoxine and trimethoprim co-exposures at different environmentally relevant concentrations on microalgal growth and nutrient assimilation.

In aquaculture around the world, sulfamonomethoxine (SMM), a long-acting antibiotic that harms microalgae, is widely employed in combination with trimethoprim (TMP), a synergist. However, their combined toxicity to microalgae under long-term exposures at environmentally relevant concentrations remains poorly understood. Therefore, we studied the effects of SMM single-exposures and co-exposures (SMM:TMP=5:1) at concentrations of 5 µg/L and 500 µg/L on Chlorella pyrenoidosa within one aquacultural drainage cycle (15 days). Photosynthetic activity and N assimilating enzyme activities were employed to evaluate microalgal nutrient assimilation. Oxidative stress and flow cytometry analysis for microalgal proliferation and death jointly revealed mechanisms of inhibition and subsequent self-adaptation. Results showed that exposures at 5 µg/L significantly inhibited microalgal nutrient assimilation and induced oxidative stress on day 7, with a recovery to levels comparable to the control by day 15. This self-adaptation and over 95 % removal of antibiotics jointly contributed to promoting microalgal growth and proliferation while reducing membrane-damaged cells. Under 500 µg/L SMM single-exposure, microalgae self-adapted to interferences on nutrient assimilation, maintaining unaffected growth and proliferation. However, over 60 % of SMM remained, leading to sustained oxidative stress and apoptosis. Remarkably, under 500 µg/L SMM-TMP co-exposure, the synergistic toxicity of SMM and TMP significantly impaired microalgal nutrient assimilation, reducing the degradation efficiency of SMM to about 20 %. Consequently, microalgal growth and proliferation were markedly inhibited, with rates of 9.15 % and 17.7 %, respectively, and a 1.36-fold increase in the proportion of cells with damaged membranes was observed. Sustained and severe oxidative stress was identified as the primary cause of these adverse effects. These findings shed light on the potential impacts of antibiotic mixtures at environmental concentrations on microalgae, facilitating responsible evaluation of the ecological risks of antibiotics in aquaculture ponds.

Recovery of nutrients from fish sludge to enhance the growth of microalga Chlorella sorokiniana CMBB276.

Intensive aquaculture production generates large amounts of sludge. This waste could be considered as a potential source of nutrients that can be recovered and utilized. Little attention has been paid to nutrient recovery from fish sludge. In this study, bioconversion of sludge was evaluated in lab scale under anaerobic (AN), facultative anaerobic (FA) and aerobic (AE) conditions. After 40 days of fermentation, AN recovered the highest values of dissolved total nitrogen (82.7 mg L-1), while AE showed the highest dissolved total phosphorus (11.8 mg L-1) and the highest reduction of total suspended solids (36.0 %). Microbial analysis showed that AN exhibited a distinct bacterial community than that of FA and AE. Furthermore, C. sorokiniana grown in AN effluents collected after 12 days of fermentation achieved the highest biomass production (1.96 g L-1). These results suggest that AN has the best potential to recover nutrients from sludge for production of C. sorokiniana.

The growth and nutrient removal properties of heterotrophic microalgae Chlorella sorokiniana in simulated wastewater containing volatile fatty acids.

To reduce the high cost of organic carbon sources in waste resource utilization in the cultivation of microalgae, volatile fatty acids (VFAs) derived from activated sludge were used as the sole carbon source to culture Chlorella sorokiniana under the heterotrophic cultivation. The addition of VFAs in the heterotrophic condition enhanced the total nitrogen (TN) and phosphorus (TP) removal of C. sorokiniana, which proved the advantageous microalgae in using VFAs in the heterotrophic culture after screening in the previous study. To discover the possible mechanism of nitrogen and phosphorus adsorption in heterotrophic conditions by microalgae, the effect of different ratios of VFAs (acetic acid (AA): propionic acid (PA): butyric acid (BA)) on the nutrient removal and growth properties of C. sorokiniana was studied. In the 8:1:1 group, the highest efficiency (77.19%) of VFAs assimilation, the highest biomass (0.80 g L-1) and lipid content (31.35%) were achieved, with the highest TN and TP removal efficiencies of 97.44 % and 91.02 %, respectively. Moreover, an aerobic denitrifying bacterium, Pseudomonas, was determined to be the dominant genus under this heterotrophic condition. This suggested that besides nitrate uptake and utilization by C. sorokiniana under the heterotrophy, the conduct of the denitrification process was also the main reason for obtaining high nitrogen removal efficiency.

A whole process study of dual microalgae cultivation coupled to domestic wastewater treatment and wheat growth.

The multiple microalgal collaborative treatment of domestic wastewater has been extensively investigated, but its whole life cycle tracking and consequent potential have not been fully explored. Herein, a dual microalgal system was employed for domestic wastewater treatment, tracking the variation in microalgal growth and pollutants removal from shake flask scale to 18 L photobioreactors scales. The results showed that Chlorella sp. HL and Scenedesmus sp. LX1 combination had superior growth and water purification performance, and the interspecies soluble algal products promoted their growth. Through microalgae mixing ratio and inoculum size optimized, the highest biomass yield (0.42 ± 0.03 g/L) and over 91 % N, P removal rates were achieved in 18 L photobioreactor. Harvested microalgae treated in different forms all promoted wheat growth and suppressed yellow leaf rate. This study provided data support for the whole process tracking of dual microalgal system in treating domestic wastewater and improving wheat growth.

A Comparative Analysis Assessing Growth Dynamics of Locally Isolated Chlorella sorokiniana and Chlorella vulgaris for Biomass and Lipid Production with Biodiesel Potential.

Prior research has emphasized the potential of microalgae in biodiesel production, driven by their ability to replace fossil fuels. However, the significant costs associated with microalgae cultivation present a major obstacle to scaling up production. This study aims to develop an eco-friendly microalgae cultivation system by integrating carbon dioxide from flue gas emissions with an affordable photobioreactor, providing a sustainable biomass production. The research evaluates the growth performance of Chlorella sorokiniana and Chlorella vulgaris across this integrated system for biomass and lipid production. Results indicate substantial biomass yields of 1.97 and 1.84 g/L, with lipid contents of 35 % and 41 % for C. sorokiniana and C. vulgaris, respectively. The macrobubble photobioreactor demonstrates high potential for microalgae biomass and lipid production, yielding quality fatty acid methyl esters such as palmitic, linoleic and stearic. This study presents an environmentally friendly system for efficient microalgae cultivation, generating lipid-rich biomass suitable for biodiesel production.

The impact of nTiO2 and GO (graphene oxide), and their combinations, on freshwater Chlorella sp.: a comparative study in lake water and BG-11 media.

Titanium dioxide nanoparticles (nTiO2) and graphene oxide (GO) are extensively used nanomaterials in various products and applications. Freshwater ecosystems are a crucial sink for these pollutants, posing severe threats to aquatic organisms. Although multiple studies have investigated the pristine toxicity of nTiO2 and GO in freshwater organisms, the combined toxicity of these materials remains unexplored. Interaction media is a crucial factor in evaluating toxicity nanomaterial toxicity towards algae. In this study, we have investigated the comparative effect of sterilized and filtered freshwater and BG-11 medium on the pristine and combined toxicity of nTiO2 and GO on freshwater algae Chlorella sp. Results indicated that the combination of nTiO2 and GO showed more toxicity when compared to their respective pristine forms. This could be due to the additive effect exhibited by nTiO2 and GO on Chlorella sp. The enhanced growth inhibition for the combined toxicity was in the order of 1 mg L-1 nTiO2 + 1 mg L-1 GO > 1 mg L-1 nTiO2 + 0.1 mg L-1 GO > 0.1 mg L-1 nTiO2 + 1 mg L-1 GO > 0.1 mg L-1 nTiO2 + 0.1 mg L-1 GO. All test groups that interacted in BG-11 media exhibited less toxicity when compared to corresponding groups in the lake water medium. This could be attributed to the cushioning effect of BG-11 medium, providing supplementary nutrition to the algal cells. This signifies that the environmentally relevant conditions could be more detrimental than the laboratory conditions. This study elucidates valuable insights into the potential detrimental effects associated with the combination of nTiO2 and GO on freshwater algae. Furthermore, we have evaluated the growth inhibition, oxidative stress, and photosynthetic activity of Chlorella sp. in both environmentally relevant interaction medium and well-defined culture medium.

Cultivation of the macrophyte Lemna minor and the microalgae Chlorella sorokiniana in thermal mineral waters: Biomass characteristics, radioisotopes and heavy metals content.

Microalgae and macrophytes are commonly used as human and animal food supplements. We examined the cultivation of the microalgae Chlorella sorokiniana and the duckweed Lemna minor in thermal waters under batch and sequencing batch conditions and we characterized the produced biomass for the presence of essential nutrients as well as for heavy metals and radioisotope content. The highest specific growth rate for the microalgae was observed when 5 or 15 mg/L N were supplemented while the optimal conditions for Lemna minor were observed in the co-presence of 5 mg/L N and 1.7 mg/L P. Lemna minor presented higher concentrations of proteins and lipids comparing to the studied microalgae. Both organisms contained high amounts of lutein (up to 1378 mg/kg for Lemna minor) and chlorophyll (up to 1518 mg/kg for Lemna minor) while ß-carotene and tocopherols were found at lower concentrations, not exceeding a few tens of mg/kg. The heavy metal content varied between the two species. Lemna minor accumulated more Cd, Cu, K, Mn, Na, Ni, and Zn whereas Al, Ca and Mg were higher in Chlorella sorokiniana. Both organisms could be a significant source of essential metals but the occasional exceedance of the statutory levels of toxic metals in food products raises concern for potential risk to either humans or animals. Application of gamma-spectroscopy to quantify the effective dose to humans from 228Ra, 226Ra and 40K showed that Chlorella sorokiniana was well under the radiological limits while the collected mass of Lemna minor was too small for radiological measurements with confidence.

Self-flocculation behaviour of cellulose-based bioflocculant synthesized from sewage water grown Chlorella sorokiniana and Scenedesmus abundans.

The global energy crisis has spurred a shift from conventional to clean and sustainable energy sources. Biomass derived from microalgae is emerging as an alternative energy source with diverse applications. Despite the numerous advantages of microalgae, large-scale biomass harvesting is not economical and convenient. Self-flocculation is considered an effective phenomenon facilitated by extracting the flocculating substances from microalgae that assist aggregation of algal cells into flocs. A novel cellulose-based bioflocculant has been synthesized from sewage water grown Chlorella sorokiniana and Scenedesmus abundans for harvesting application. The produced bioflocculant amounted to 38.5% and 19.38% of the dry weight of S. abundans and C. sorokiniana, respectively. Analysis via FTIR, XRD, and FESEM-EDX revealed the presence of cellulose hydroxyapatite (HA) in algae-derived cellulose. Harvesting efficiencies of 95.3% and 89.16% were attained for S. abundans and C. sorokiniana, respectively, at a dosage of 0.5 g/L. Furthermore, the bioflocculant was recovered, enabling its reuse with recovery efficiencies of 52% and 10% for S. abundans and C. sorokiniana, respectively. This simple and efficient approach has the potential to replace other harvesting methods, thereby contributing to the economic algal biofuel production.

Growth, nutrient removal, and lipid productivity promotion of Chlorella sorokiniana by phosphate solubilizing bacteria Bacillus megatherium in swine wastewater: Performances and mechanisms.

Effects of a phosphorus-solubilizing bacteria (PSB) Bacillus megatherium on growth and lipid production of Chlorella sorokiniana were investigated in synthesized swine wastewater with dissolved inorganic phosphorus (DIP), insoluble inorganic phosphorus (IIP), and organic phosphorus (OP). The results showed that the PSB significantly promoted the algal growth in OP and IIP, by 1.10 and 1.78-fold, respectively. The algal lipid accumulation was also greatly triggered, respectively by 4.39, 1.68, and 1.38-fold in DIP, IIP, and OP. Moreover, compared with DIP, OP improved the oxidation stability of algal lipid by increasing the proportion of saturated fatty acids (43.8 % vs 27.9 %), while the PSB tended to adjust it to moderate ranges (30.2-41.6 %). Further, the transcriptome analysis verified the OP and/or PSB-induced up-regulated genes involving photosynthesis, lipid metabolism, signal transduction, etc. This study provided novel insights to enhance microalgae-based nutrient removal combined with biofuel production in practical wastewater, especially with complex forms of phosphorus.

Continuous microalgal culture module and method of culturing microalgae containing macular pigment.

This study established and investigated continuous macular pigment (MP) production with a lutein (L):zeaxanthin (Z) ratio of 4-5:1 by an MP-rich Chlorella sp. CN6 mutant strain in a continuous microalgal culture module. Chlorella sp. CN6 was cultured in a four-stage module for 10 days. The microalgal culture volume increased to 200 L in the first stage (6 days). Biomass productivity increased to 0.931 g/L/day with continuous indoor white light irradiation during the second stage (3 days). MP content effectively increased to 8.29 mg/g upon continuous, indoor white light and blue light-emitting diode irradiation in the third stage (1 day), and the microalgal biomass and MP concentrations were 8.88 g/L and 73.6 mg/L in the fourth stage, respectively. Using a two-step MP extraction process, 80 % of the MP was recovered with a high purity of 93 %, and its L:Z ratio was 4-5:1.

Developing a Chromochloris zofingiensis Mutant for Enhanced Production of Lutein under CO2 Aeration.

Microalgae are competitive and commercial sources for health-benefit carotenoids. In this study, a Chromochloris zofingiensis mutant (Cz-pkg), which does not shut off its photosystem and stays green upon glucose treatment, was generated and characterized. Cz-pkg was developed by treating the algal cells with a chemical mutagen as N-methyl-N'-nitro-N-nitrosoguanidine and followed by a color-based colony screening approach. Cz-pkg was found to contain a dysfunctional cGMP-dependent protein kinase (PKG). By cultivated with CO2 aeration under mixotrophy, the mutant accumulated lutein up to 31.93 ± 1.91 mg L-1 with a productivity of 10.57 ± 0.73 mg L-1 day-1, which were about 2.5- and 8.5-fold of its mother strain. Besides, the lutein content of Cz-pkg could reach 7.73 ± 0.52 mg g-1 of dry weight, which is much higher than that of marigold flower, the most common commercial source of lutein. Transcriptomic analysis revealed that in the mutant Cz-pkg, most of the genes involved in the biosynthesis of lutein and chlorophylls were not down-regulated upon glucose addition, suggesting that PKG may regulate the metabolisms of photosynthetic pigments. This study demonstrated that Cz-pkg could serve as a promising strain for both lutein production and glucose sensing study.

Bioremediation of hemotoxic and oxidative stress induced by polyethylene microplastic in Clarias gariepinus using lycopene, citric acid, and chlorella.

Despite extensive research on the toxic effects of microplastics (MPs), there is no obtainable data on the use of phytobioremediation against MPs toxicity in fish. This study aimed to investigate the protective role of lycopene, citric acid, and chlorella against the toxic effects of MPs in African catfish (Clarias gariepinus) using hematology, biochemical, antioxidants, erythron profiles (poikilocytosis and nuclear abnormalities) and the accumulation of MPs in tissues as biomarkers. Five groups of fish received: normal diet (control); MPs (500 mg/kg diet) (Group 2); MPs (500 mg/kg diet) + lycopene (500 mg/kg diet) (Group 3); MPs (500 mg/kg diet) + citric acid (30 g/kg diet) (Group 4); and MPs (500 mg/kg diet) + chlorella (50 g/kg diet) (Group 5) for 15 days. Group 2 had significantly higher amounts of MPs in the stomach, gills, and feces, electrolyte imbalances (HCO3, Fe, Na+, K+, Ca+2, Cl-, and anion gap, hematobiochemical alterations, and decreases in the activities of superoxide dismutase, catalase, total antioxidant capacity, and glutathione S-transferases compared to the control group. Additionally, Group 2 had significant increase in the percentage of poikilocytosis, and nuclear abnormalities in RBC's compared to the control group. The co-treatment of MPs-exposed fish with lycopene, citric acid, and chlorella-supplemented diets ameliorated the hematological, biochemical, and erythron profile alterations, but only slightly enhanced the antioxidant activity. Overall, lycopene, citric acid, and chlorella can be recommended as a feed supplement to improve hematobiochemical alterations and oxidative damage induced by MPs toxicity in the African catfish (C. gariepinus).

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production.

Heterotrophic cultivation of Chlorella has achieved commercial success, but the application of Chlorella biomass is still limited due to the high cost of biomass production. In this study, an effective and industrially scalable heterotrphic cultivation technology has been developed for a production strain Chlorella sorokiniana GT-1. Under the optimized culturing conditions, the ultrahigh biomass concentration of 271 and 247 g L-1 was achieved in 7.5 L bench-scale and 1000 L pilot-scale fermenters, respectively. Technoeconomic (TE) analysis indicated that the production cost of C. sorokiniana GT-1 could be reduced to $1601.27 per ton of biomass if the biomass concentration reached 200 g L-1 , which is 24.2% lower than that of the reported highest Chlorella biomass production through fermentation with the same TE model. Under the same growth conditions, the maximum biomass concentration of a low-starch mutant SLM2 was reduced to 93 g L-1 , which was 54% lower than that of the wild type, indicating the capabilities of C. sorokiniana GT-1 cells in accumulating large amounts of starch are essential for achieving the ultrahigh-cell-density under the heterotrophic conditions. In addition, the ultrahigh-cell-density growth potential of C. sorokiniana GT-1 cells was inferred to be related to the intrinsic biological characteristics including the tolerance to low dissolved oxygen and a moderate doubling time under the heterotrophic conditions as well. The breakthrough in cultivation technology is promising for Chlorella industry and would expand its applications in food and feed.

Efficient isolation and cultivation of endosymbiotic Chlorella from Paramecium bursaria on agar plates by co-culture with yeast cells.

Paramecium bursaria is a ciliate that harbors Chlorella-like unicellular green algae as endosymbionts. The relationship between the host P. bursaria and the endosymbiotic Chlorella is facultative; therefore, both partners can be cultured independently and re-combined to re-establish symbiosis, making this system suitable for studying algal endosymbiosis. However, despite many previous studies, cultivation of endosymbiotic Chlorella remains difficult, particularly on agar plates. Here we describe a simple agar plate method for efficiently isolating and culturing cells of the endosymbiotic alga Chlorella variabilis from an individual P. bursaria cell, by co-culturing them with yeast Saccharomyces cerevisiae. The co-culture with the yeast significantly improved the colony-forming efficiency of the alga on agar. Growth assays suggest that the main role of the co-cultured yeast cells is not to provide nutrients for the algal cells, but to protect the algal cells from some environmental stresses on the agar surface. Using the algal cells grown on the plates and a set of specially designed primers, direct colony PCR can be performed for screening of multiple endosymbiont clones isolated from a single host ciliate. These methods may provide a useful tool for studying endosymbiotic Chlorella species within P. bursaria and various other protists.