Astaxanthin Production Research Articles

Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts

The slow growth and complex life cycle of Haematococcus lacustris pose significant challenges for cost-effective astaxanthin production. This study explores the use of microfluidic collision treatment to stimulate the germination of dormant seed cysts, thereby improving photosynthetic cell growth and astaxanthin productivity in H. lacustris cultivated in well plate and flask cultures. The flow rate (1.0–3.0 mL/min) and the number of T-junction loops (3–30) were optimized in the microfluidic device. Under optimal conditions (a flow rate of 2.0 mL/min with 10 loops), the total cell number density in well plate cultures increased by 44.5% compared to untreated controls, reaching 28.9 ± 2.0 × 104 cells/mL after 72 h. In flask cultures, treated cysts showed a 21% increase in astaxanthin productivity after 30 d, reaching 0.95 mg/L/d, due to higher biomass concentrations, while the astaxanthin content per cell remained constant. However, excessive physical collision stress at higher flow rates and loop numbers resulted in reduced cell viability and cell damage. These findings suggest that carefully controlled cyst mechanostimulation can be an effective and environmentally friendly strategy for Haematococcus biorefining, enabling the production of multiple bioactive products.

Two-stage carbon sequestration by Haematococcus pluvialis: Integrated research from small-scale to pilot-scale cultivation and data quality monitoring.

A novel two-stage carbon sequestration strategy (3 % and 10 % CO2) was developed and its feasibility was comprehensively demonstrated by multiple methods (pilot-scale cultivation, kinetics, economics and carbon fixation analysis). It was also a safe, efficient and low-cost harvesting strategy. At the end of the culture, astaxanthin production and content increased 2.3 and 2.2 times, respectively. Sedimentation rate (SR) was introduced for the first time to evaluate microalgae culture methods. The SR reached 82.2 % after 2 h of standing. Pilot-scale cultivation was achieved outdoors, with the optimal photobioreactor being a 40 L tubular photobioreactor (T-PBRs), which individually achieved 3.1 g/L and 2.3 % biomass and astaxanthin content. The maximum rate of carbon sequestration (227.9 mg/L/d) was observed in 40 L T-PBRs. The cost of producing 1 kg of astaxanthin-enriched Haematococcus pluvialis (H. pluvialis) was only 17.5 USD. This study brings new perspectives to carbon sequestration and the development of astaxanthin markets.

Regulation Mechanism of Gibberellic Acid-3 for Astaxanthin Biosynthesis in Heterotrophic Growing Chromochloris zofingiensis.

Chromochloris zofingiensis has been proven as a potential resource for large-scale astaxanthin production, but little information on phytohormones for its growth and astaxanthin accumulation could be obtained. This study explored the impact of gibberellic acid-3 (GA3) on growth and astaxanthin biosynthesis in heterotrophic C. zofingiensis. After 6 days of cultivation with GA3, biomass and astaxanthin yields in 7.5 L fermenters reached 268.5 g·L-1 and 0.34 g·L-1, respectively, which were 6% and 89% higher than those in the control. GA3 changed transcription levels of genes linked to carbon metabolism, lipid metabolism, astaxanthin production, and ABC transporters. Genes related to astaxanthin biosynthesis, such as phytoene synthase (PSY), phytoene desaturase (PDS), beta-carotenoid hydroxylase (CHYb), and beta-carotenoid ketolase (BKT), were up-regulated under GA3 induction. The enhancement of carbon metabolism and lipid metabolism led to elevated consumption of substrates and generation of reducing power, thus facilitating astaxanthin biosynthesis. By using GA3 and arginine together, the astaxanthin yield increased to 0.39 g·L-1, which was 18% higher than that obtained under GA3 induction. It could be concluded that GA3 showed significant effects on astaxanthin biosynthesis. This research proposed novel feasible approaches to enhance astaxanthin production in heterotrophic C. zofingiensis.

Metabolic engineering of Mucor circinelloides to improve astaxanthin production.

Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72h and a 5.5-fold increase at 24h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57g/L compared to 9.1 ± 0.23g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides.

Production of Carotenoids and Astaxanthin from Haematococcus pluvialis Cultivated Under Mixotrophy Using Brewery Wastewater: Effect of Light Intensity and Cultivation Time

Production of Carotenoids and Astaxanthin from Haematococcus pluvialis Cultivated Under Mixotrophy Using Brewery Wastewater: Effect of Light Intensity and Cultivation Time

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for itsstrong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application inmedicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longercompared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged asa promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use ofthe natural host Xanthophyllomyces dendrorhous, andthe heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

Enhancement of ketocarotenoid production using the heterologous orange protein from Ipomoea batatas in indigenous microalga Ettlia sp.

Astaxanthin, one of the most powerful natural antioxidants, is used in high-value industries such as those of cosmetics, nutraceuticals, and food products derived from microalgae. In this study, Ettlia sp. mutants were generated by expressing two types of heterologous orange proteins, IbOr and IbOr-R96H, in which the 96th arginine of IbOr was substituted with histidine derived from Ipomoea batatas to enhance astaxanthin production. The Ett-IbOr-R96H mutant showed a 2.4-fold increase in β-carotene content compared to the wildtype (4.59 mg g−1DCW), reaching 10.82 mg g−1 under high-light conditions via two-phase cultivation. Under the stress treatment combination of high-light intensity and nitrogen deprivation, total carotenoid content increased to 17.24 mg L−1 and 21.94 mg L−1 in Ett-IbOr and Ett-IbOr-R96H, respectively. The astaxanthin and canthaxanthin contents in Ett-IbOr-R96H was 4.89 mg L−1 and 0.47 mg L−1, respectively, which were 1.8- and 1.5-fold higher, respectively, than those in Ett-IbOr. Additionally, photosynthetic efficiency (Fv/Fm) recovered in Ett-IbOr-R96H under dual-stress conditions compared to the wildtype while reactive oxygen species levels decreased throughout the cultivation period. Our findings suggest that the heterologous IbOr expression in Ettlia sp. may be an effective approach for enhancing the production of ketocarotenoids and improving stress resistance for industrial applications.

Novel insights into saline stress on photosynthetic activity and astaxanthin production of Haematococcus pluvialis

Novel insights into saline stress on photosynthetic activity and astaxanthin production of Haematococcus pluvialis

Gamma-aminobutyric acid as a regulator of astaxanthin production in Haematococcus lacustris under salinity: Exploring physiology, signaling, autophagy, and multi-omics landscape

Haematococcus lacustris-derived natural astaxanthin has significant commercial value, but stressful conditions alone impair cell growth and reduce the total productivity of astaxanthin in industrial settings. This study used gamma-aminobutyric acid (GABA) to increase biomass, astaxanthin productivity, and tolerance to salinity. GABA under NaCl stress enhanced the biomass to 1.76 g/L, astaxanthin content to 30.37 mg g−1, and productivity to 4.10 mg/L d−1, outperforming the control. Further analysis showed GABA enhanced nitrogen assimilation, Ca2+ level, and cellular GABA content, boosting substrate synthesis, energy metabolism, osmoregulation, autophagy, and antioxidant defenses. GABA also activated signaling pathways involving phytohormones, cAMP, cGMP, and MAPK, aiding astaxanthin synthesis. The application of biomarkers (ethylene, salicylic acid, trans-zeatin) and an autophagy inhibitor cooperated with GABA to further enhance the total astaxanthin productivity under NaCl stress. Combining GABA with 25 μM salicylic acid maximized astaxanthin yield at 4.79 mg/L d−1, offering new strategies for industrial astaxanthin production.

Study on the effect of benomyl on the ability to enhance astaxanthin production of Rhodosporidium toruloidesand evaluate the free radical scavenging activity ABTS of astaxanthin

Study on the effect of benomyl on the ability to enhance astaxanthin production of Rhodosporidium toruloidesand evaluate the free radical scavenging activity ABTS of astaxanthin

Enhanced astaxanthin production in Haematococcus lacustris by electrochemical stimulation of cyst germination

This study investigated the technical feasibility of using electrogermination to activate dormant cysts as an inoculum for subsequent 14-d photosynthetic astaxanthin production in Haematococcus lacustris. Electrotreatment affected the cell viability, surface charge, and morphology of H. lacustris cysts. At an optimal voltage of 2 V for 60 min, the cyst germination rate peaked at 44.6 % after 1 d, representing a 2.2-fold increase compared with that of the untreated control. Notably, electrogermination significantly enhanced both the astaxanthin content (44.9 mg/g cell) and productivity (13.2 mg/L/d) after 14 d of photobioreactor cultivation, corresponding to 1.7- and 1.5-fold increases compared with those in control, respectively. However, excessive electrotreatment, particularly at voltages exceeding 2 V or for durations beyond 60 min, did not enhance the astaxanthin production capability of H. lacustris. Proper optimization of renewable electrogermination can enable sustainable algal biorefinery to produce multiple bioactive products without compromising cell viability and astaxanthin productivity.

Screening and Selection of Antibiotics for Enhanced Production of Astaxanthin by Haematococcus lacustris.

Haematococcus lacustris (Girod-Chantrans) Rostafinski (Chlorophyta) is the richest microalgal source of astaxanthin. Natural astaxanthin from H. lacustris has been widely studied and used for commercial production worldwide. In this study, we examined the effects of 11 antibiotics (dihydrostreptomycin sulphate, neomycin, chloramphenicol, penicillin, streptomycin, ampicillin, kanamycin, gentamycin, hygromycin B, tetracycline, and paromomycin) on the biomass dry weight, growth, and astaxanthin yield of H. lacustris using Jaworski's medium without a nitrogen source. Astaxanthin content in H. lacustris was improved in the presence of ampicillin (0.25 g/L, 0.5 g/L, 1 g/L), chloramphenicol (0.25 g/L), and penicillin (0.25 g/L, 0.5 g/L, 1 g/L) in comparison to the control on day 15. The greatest increase in astaxanthin content on day 15 (6.69-fold) was obtained with the addition of penicillin (0.5 g/L) in comparison to the control. Similarly, on day 15, the cell numbers were also the highest for the H. lacustris culture grown with the addition of penicillin (0.5 g/L).

Insight into the regulation mechanism of light spectra on astaxanthin biosynthesis in mixotrophic Chromochloris zofingiensis

This study studied the effects of white light (WL), blue light (BL), and yellow-green light (YGL) on astaxanthin production in mixotrophically grown Chromochloris zofingiensis. The biomass yield achieved under BL was higher than that obtained under WL and YGL, respectively. Remarkably, compared with WL and YGL, the RuBisCO activity under BL was reduced, suggesting that the light wavelength could influence the RuBisCO activity. The results showed that BL was more suitable for astaxanthin accumulation than WL and YGL. When C. zofingiensis was grown under BL, the total yields of astaxanthin reached 0.2844 g·L−1 in 12 days, of which the biomass yield was 204.5 g·L−1 and astaxanthin content was 0.139 % of DW. The metabolomic study revealed that the central carbon metabolism and astaxanthin biosynthesis were enhanced, supporting fast cell growth and high astaxanthin contents under BL. Moreover, BL could enhance astaxanthin production by increasing the PSY, LCYb, CHYb, and BKT gene expression. This study provides effective strategies to facilitate astaxanthin production in C. zofingiensis.

Biovalorisation of agro-industrial wastes into astaxanthin by Xanthophyllomyces dendrorhous

Astaxanthin is a red xanthophyll with high economic and industrial value in the pharmaceutical, nutraceutical, cosmetic and food industries. In recent years, the biotechnological production of astaxanthin has attracted much attention as a sustainable alternative to the predominating petrochemical-dependent chemical synthesis. In this regard, Xanthophyllomyces dendrorhous is regarded as a promising microorganism for industrial production of astaxanthin. Unfortunately, biotechnological production of the carotenoid is currently expensive. The present study investigated soy molasses (SM) and residual brewers’ yeast as cheap fermentation feedstocks for the cultivation of X. dendrorhous and astaxanthin production. Yeast extract was obtained from residual brewers’ yeast using various techniques and then combined with SM to formulate a two-component growth medium which was subsequently used to cultivate X. dendrorhous. Generally, the yeast extract produced from residual brewers’ yeast supported X. dendrorhous growth and astaxanthin production at levels comparable to those seen with commercial yeast extract. Overall, cultivating X. dendrorhous in an SM-based medium containing 5% SM and 0.2% yeast extract obtained from residual brewers’ yeast resulted in significantly higher (> 20% more) biomass accumulation compared to the control media (YPD). A similar slightly higher astaxanthin output (up to 14% more) was recorded in the SM-based medium compared to YPD. The formulated cultivation medium in this study provides an opportunity to reduce the production cost of astaxanthin from X. dendrorhous while simultaneously reducing the environmental impact related to the disposal of the industrial waste used as feedstock.Key points• Cheap culture media were formulated from soy molasses and brewers’ spent yeast• The formulated medium resulted in at least 20% more biomass than the control• Up to 14% more astaxanthin was produced in molasses-based mediumGraphical

Utilization of silicon dioxide nanoparticles and silicon salts to enhance astaxanthin production in Haematococcus Pluvialis

Haematococcus pluvialis is a type of microalgae that is commercially important as it is the primary source of natural astaxanthin - a potent antioxidant used in nutraceuticals, cosmetics, food, and aquaculture industries. Various nanoparticles and chemicals have been used to stimulate the growth of H. pluvialis to increase astaxanthin production. In this study, silicon nanoparticles were synthesized from tetraethyl orthosilicate (TEOS) and characterized to ensure the pure synthesis of uniform nanocrystals of silicon dioxide. The microalgae were cultivated in optimal Haematococcus medium (OHM) under specific conditions, including a temperature of 25 °C, a pH of 7, and a light intensity of 50 μmol. s−1.m−2 for 12 h of light and 12 h of darkness. To study the amount of viability and astaxanthin production, the microalgae were exposed to different concentrations of silicon, sodium silicate, calcium silicate, and potassium silicate. The highest astaxanthin production (194 mg/g) was obtained after stimulation at 200 μg/ml of silicon compared to the control after 15 days by high-performance liquid chromatography (HPLC) technique. Therefore, it can be concluded that certain concentrations of silicon and silicon salts can be considered a suitable stimulus to produce astaxanthin in the H. pluvialis microalgae. This study highlights the potential of using silicon nanoparticles as a stimulant for astaxanthin production in H. pluvialis, which could have significant implications for the nutraceuticals, cosmetics, food, and aquaculture industries.

Preliminary assessment of astaxanthin production in a new Chlamydomonas strain

Green microalgae are increasingly valuable in industries such as food, cosmetics, animal feed, functional foods, and pharmaceuticals due to their ability to produce significant secondary metabolites like carotenoid pigments. Despite the growing demand for microalgae-derived carotenoids, the identification of robust wild-type strains with high biomass productivity under specific growth conditions remains limited. This study introduces Chlamydomonas sp. KIOST-2 (accession number: PP532860), a newly identified wild-type microalgal strain with 99.9 % genetic similarity to Chlamydomonas callosa, characterized through 18S rRNA gene sequence analysis. Notably, Chlamydomonas sp. KIOST-2 demonstrates considerable biomass productivity at 20–30 °C under alkaline (pH 8–10) and freshwater conditions, making it suitable for large-scale cultivation. Under drought stress, this strain forms orange cysts with high concentrations of astaxanthin (5.7 ± 0.6 mg/g) and notable lipid accumulation, primarily of oleic acid (C18:1 n9c), palmitic acid (C16:0), and linoleic acid (C18:2 n6c). The ability of Chlamydomonas sp. KIOST-2 to produce substantial amounts of astaxanthin under drought conditions without genetic modification highlights its potential for biorefinery applications and industrial exploitation. This discovery underscores the strain's unique combination of drought resistance and high astaxanthin productivity, positioning it as a valuable source of bioactive compounds for various industries.

Comparative genomic analysis and optimization of astaxanthin production of Rhodotorula paludigena TL35-5 and Rhodotorula sampaioana PL61-2.

Astaxanthin is a powerful antioxidant known to enhance skin, cardiovascular, eye, and brain health. In this study, the genome insights and astaxanthin production of two newly isolated astaxanthin-producing yeasts (TL35-5 and PL61-2) were evaluated and compared. Based on their phenotypic and genotypic characteristics, TL35-5 and PL61-2 were identified as basidiomycetous yeasts belonging to Rhodotorula paludigena and Rhodotorula sampaioana, respectively. To optimize astaxanthin production, the effects of cultural medium composition and cultivation conditions were examined. The optimal conditions for astaxanthin production in R. paludigena TL35-5 involved cultivation in AP medium containing 10 g/L glucose as the sole carbon source, supplemented with 1.92 g/L potassium nitrate, pH 6.5, and incubation at 20°C for 3 days with shaking at 200 rpm. For R. sampaioana PL61-2, the optimal medium composition for astaxanthin production consisted of AP medium with 40 g/L glucose, supplemented with 0.67 g/L urea, pH 7.5, and the fermentation was carried out at 20°C for 3 days with agitating at 200 rpm. Under their optimal conditions, R. paludigena TL35-5 and R. sampaioana PL61-2 gave the highest astaxanthin yields of 3.689 ± 0.031 and 4.680 ± 0.019 mg/L, respectively. The genome of TL35-5 was 20,982,417 bp in length, with a GC content of 64.20%. A total of 6,789 protein-encoding genes were predicted. Similarly, the genome of PL61-2 was 21,374,169 bp long, with a GC content of 64.88%. It contained 6,802 predicted protein-encoding genes. Furthermore, all essential genes involved in astaxanthin biosynthesis, including CrtE, CrtYB, CrtI, CrtS, and CrtR, were identified in both R. paludigena TL35-5 and R. sampaioana PL61-2, providing evidence for their ability to produce astaxanthin.

Sustainable conversion of carbon dioxide to high-value antioxidant astaxanthin through microbial electrosynthesis-assisted microalgae cultivation

Microbial electrosynthesis (MES) effectively converts carbon dioxide (CO2) to short carboxylic acids but has lower efficiency for higher molecular value-added products. This study used a two-stage process integrating MES with microalgae cultivation to convert CO2 into a value-added chemical—astaxanthin, a xanthophyll carotenoid with strong antioxidant activity. In the first stage, MESs featuring platinum-coated anodes and carbonaceous cathodes were employed to bioelectrochemically convert CO2 to volatile fatty acids (VFAs). In the second stage, microalgal cultivation for sustainable astaxanthin production was conducted using a VFA-containing MES effluent. In the MES operation, a newly designed cathode with alternating layers of carbon felt (CF) and reticulated vitreous carbon (RVC) significantly improved hydrodynamics and bacterial attachment. Consequently, the CF/RVC composite cathode produced 7.68 g/L VFAs from CO2, with the highest production rate of 1225.72 mg/L/day and the highest coulombic efficiency of 94.15 % at 2.8 V. Feeding acetate-containing catholyte effluent demonstrated faster growth of microalgae and astaxanthin accumulation compared to autotrophic culture, indicating synergistic integration of MES and microalgae for capturing value from CO2. Among tested microalgae strains, Haematococcus pluvialis most efficiently accumulated astaxanthin up to 12.95 mg/L as cultured with MES effluent. Successful CO2-to-astaxanthin conversion using MES and microalgae can contribute to resolving unprecedented environmental challenges attributed to the excess emission of CO2.

Sequential two-stage cultivation system using novel microalga consortia for treatment of municipal wastewater and simultaneous biomass production: Sustainable environmental management

Monoculture-based microalgae cultivation systems to treat wastewater are well-reported. Despite that, this method has some limitations in terms of nutrient removal potential, environment adaptation, and low biomass productivity. Conversely, microalgae co-cultivation and a two-stage sequential cultivation system (TSSCS) recently emerged as a promising approach to improve the treatment process and biomass productivity through better adaptation to the environment. However, no outdoor large-scale experiments were reported using this approach which hinders the viability of the process. Thus, in the present study, a sequential two-stage large-scale outdoor novel microalgae consortia experiment was developed. In first stage consortia-assisted sequential cultivation, two ratios of Tetraselmis indica (TS) and one ratio of Picochlorum sp. (PC) (2 TS:1 PC) were cultivated in a 1000-L pond containing 75%-municipal wastewater (MWW) + 25%-ASN-III, while in the second stage, 2 PC:1 TS was cultivated in two different ponds, and each containing 375-L 2 TS:1 PC-treated water + 375-L ASN-III. Outdoor parameters and nutrient removal efficiency (NRE), biomass, and biomolecule productivity such as lipid, photosynthetic pigments, astaxanthin, and β-carotene were quantified, and cost analysis was performed. At the end of the first and second stages, 2 TS:1 PC and 2 PC:1 TS showed maximum NRE of COD (68.71 and 86.40%), TN (66.98 and 94.73%), and TP (82.70 and 94.36%), respectively. Moreover, 2 TS:1 PC and 2 PC:1 TS Pond 1 and 2 produced maximum dry biomass production; 2.41 and ∼2.54 g/L contained lipid content; 36.89 and 34.90% that have 86.50 and 55.79% FAME content respectively. Similarly, 2 TS:1 PC and 2 PC:1 TS biomass exhibited valuable pigments production of astaxanthin i.e., 0.56 and 0.35 mg/g, and β-carotene; 4.65 and 2.82 mg/g, respectively. The cost analysis suggested that only microalgal-based MWW treatment was unfeasible, while valorization of produced biomass into co-products could offset the operation costs and could allow the option for the microalgal-based sustainable approach for the treatment of MWW and recovery of valuable resources.

Effect of carrot juice on Haematococcus pluvialis growth and astaxanthin production

In this work, we report the use of carrot juice (CJ) as a source of carotenoids as precursors of astaxanthin in a microalga Haematococcus pluvialis culture medium. Three experiments were carried out, two with the culture media supplemented with CJ (1.62 g.mL−1) at the beginning of the vegetative phase, and at the peak of the cell growth; and a control (without CJ). The samples supplemented with CJ resulted in a biomass production (531.9 mg and 625.3 mg) higher than the control (445.9 mg). Similar results were observed in astaxanthin production, resulting in a considerably higher amount (8.54 and 7.71 ppm) in the experiments supplemented with CJ compared to the control (4.79 ppm). These results showed the potential of CJ use as an additive to significatively enhance both the biomass of H. pluvialis and astaxanthin content, especially when CJ is added at the beginning of the microalga culture.