Optimization of Heterotrophic Culture Conditions for the Algae Graesiella emersonii WBG-1 to Produce Proteins.

The aim of this study was to improve the protein content and yield of heterotrophic microalgal cultivation and establish a simple, economical, and efficient method for microalgal protein production using the novel green alga, Graesiella emersonii WBG-1, which has not been previously reported for heterotrophic cultivation. Through batch heterotrophic cultivation of this alga, we observed that glucose was the optimal carbon source, while it could not use sucrose as a carbon source. Biomass production and protein content were significantly reduced when sodium acetate was used as the carbon source. Compared with nitrate, protein content increased by 93% when urea was used as the nitrogen source. Cultivation temperature had a significant impact on biomass production and protein content. The optimal conditions were glucose as the carbon source at an initial concentration of 10 g/L, urea as the nitrogen source at an initial concentration of 1.62 g/L, and a culture temperature of 35 °C. On the second day of batch cultivation, the highest protein content (66.14%) was achieved, which was significantly higher than that reported in heterotrophic cultures of Chlorella and much higher than that reported for specially established technologies aimed at increasing the protein content, such as two-stage heterotrophic, heterotrophy-dilution-photoinduction, and mixotrophic processes. These results demonstrate the great potential of the heterotrophic cultivation of G. emersonii WBG-1 for protein production.

Improving the Thermal and Oxidative Stability of Food-Grade Phycocyanin from Arthrospira platensis by Addition of Saccharides and Sugar Alcohols.

The water-soluble pigment protein phycocyanin (C-PC) from cyanobacteria Arthrospira sp. is an excellent natural food colorant and nutritional supplement with a brilliant blue color. However, C-PC is highly unstable, especially at high temperatures and when exposed to oxidative stress. The lack of simple and economical methods for improving the stability of C-PC greatly limits the application of this functional protein in the food industry. This study investigated the effect of adding saccharides (glucose, mannose, galactose, and maltose) and sugar alcohols (mannitol and maltitol) on the stability of food-grade C-PC extracted from Arthrospira platensis; the relevant reaction kinetics were also analyzed. The results revealed that glucose, mannose, mannitol, galactose, and maltose could effectively improve the thermal stability of C-PC. This improvement was positively correlated with the concentration of the additives and decreased sharply when the temperature exceeded 60 °C. Furthermore, the results also revealed the instability of C-PC when subjected to oxidative stress and the effectiveness of glucose, mannose, mannitol, and maltose in preventing the oxidative degradation of C-PC. In general, this study demonstrates that glucose, mannose, mannitol, and maltose are promising compounds for promoting the thermal and oxidative stability of C-PC, providing an economical and effective method for C-PC preservation.

Physiological and Dual Transcriptional Analysis of Microalga Graesiella emersonii-Amoeboaphelidium protococcarum Pathosystem Uncovers Conserved Defense Response and Robust Pathogenicity.

The underlying mechanisms of microalgal host-pathogen interactions remain largely unknown. In this study, we applied physiological and simultaneous dual transcriptomic analysis to characterize the microalga Graesiella emersonii-Amoeboaphelidium protococcarum interaction. Three infection stages were determined according to infection rate and physiological features. Dual RNA-seq results showed that the genes expression of G. emersonii and A. protococcarum were strongly dynamically regulated during the infection. For microalgal hosts, similar to plant defense response, the expression of defense genes involved in the pattern recognition receptors, large heat shock proteins, and reactive oxygen scavenging enzymes (glutathione, ferritin, and catalase) were significantly upregulated during infection. However, some genes encoding resistance proteins (R proteins) with a leucine-rich repeat domain exhibited no significant changes during infection. For endoparasite A. protococcarum, genes for carbohydrate-active enzymes, pathogen-host interactions, and putative effectors were significantly upregulated during infection. Furthermore, the genes in cluster II were significantly enriched in pathways associated with the modulation of vacuole transport, including endocytosis, phagosome, ubiquitin-mediated proteolysis, and SNARE interactions in vesicular transport pathways. These results suggest that G. emersonii has a conserved defense system against pathogen and that endoparasite A. protococcarum possesses a robust pathogenicity to infect the host. Our study characterizes the first transcriptomic profile of microalgae-endoparasite interaction, providing a new promising basis for complete understanding of the algal host defense strategies and parasite pathogenicity.

Controlling of two destructive zooplanktonic predators in Chlorella mass culture with surfactants.

BACKGROUND: Predatory flagellates and ciliates are two common bio-contaminants which frequently cause biomass losses in Chlorella mass culture. Efficient and targeted ways are required to control these contaminations in Chlorella mass cultivation aiming for biofuel production especially. RESULTS: Five surfactants were tested for its ability to control bio-contaminations in Chlorella culture. All five surfactants were able to eliminate the contaminants at a proper concentration. Particularly the minimal effective concentrations of sodium dodecyl benzene sulfonate (SDBS) to completely eliminate Poterioochromonas sp. and Hemiurosomoida sp. were 8 and 10 mg L-1, respectively, yet the photosynthesis and viability of Chlorella was not significantly affected. These results were further validated in Chlorella mass cultures in 5, 20, and 200 m2 raceway ponds. CONCLUSIONS: A chemical method using 10 mg L-1 SDBS as pesticide to control predatory flagellate or ciliate contamination in Chlorella mass culture was proposed. The method helps for a sustained microalgae biomass production and utilization, especially for biofuel production.

Application of surfactants for controlling destructive fungus contamination in mass cultivation of Haematococcus pluvialis.

Large-scale cultivation of Haematococcus pluvialis is frequently contaminated by the destructive fungus Paraphysoderma sedebokerense, which can cause huge losses in astaxanthin production. Here, we propose the use of four commercial surfactants to control P. sedebokerense contamination in H. pluvialis cultures. In laboratory experiments, sodium dodecylbenzene sulfonate, sodium dodecyl sulfate and primary alcohol ethoxylate treatments showed inhibitory effects on fungal contamination. Moreover, sodium dodecylbenzene sulfonate was the most promising because treatment at 7 mg L-1 was effective against fungal infection without negatively affecting the growth or astaxanthin contents of H. pluvialis. This could be ascribed to their different cell coverings and structures. Additionally, applying sodium dodecylbenzene sulfonate to open raceway ponds prevented fungal contamination, and astaxanthin production reached 1.82 g m-2. Therefore, sodium dodecylbenzene sulfonate can be used as an effective and economical control agent for commercial production of astaxanthin fromH. pluvialis.

Effects of carbon concentration, pH, and bubbling depth on carbon dioxide absorption ratio in microalgae medium.

The microalgae-based CO2 sequestration is considered to be an effective technique with great potential to cope with carbon emission. However, most researches are only focused on microalgae; the effects of physicochemical factors, which are carbon concentration, medium pH, and bubbling depth, on absorption and utilization of supplied CO2 in culture is less known. In order to understand and improve CO2 absorption in microalgae culture, the effects of these three factors were studied with different levels and combinations. Results revealed that when medium carbon concentration increased from 4.76 to 95.24 mmol/L, CO2 absorption ratio increased by about 12%, 10%, 12%, and 11% at medium depths of 10, 20, 40, and 80 cm, with the initial pH 10.6 to 9.7 by bubbling CO2, respectively. As bubbling depth increased from 10 to 80 cm, CO2 absorption ratio increased by about 25%, 22%, and 25% at carbon concentrations of 4.76, 9.52, and 95.24 mmol/L, with the initial pH 10.6 to 9.7 by bubbling CO2, respectively. In range of 10.6-7.0, pH had no significant effect on CO2 absorption ratio (P > 0.05) when carbon concentration is below 9.52 mmol/L, while above 9.52 mmol/L, pH had significant effect on CO2 absorption ratio (P < 0.05). It was found for the first time that the effect of pH on the CO2 absorption ratio was affected by carbon concentration. In addition, equilibrium pH, at which the CO2 partial pressure in the medium equals to that in the air, of medium with different carbon concentrations was also determined. Overall, in microalgae culture for CO2 sequestration, increasing CO2 bubbling depth and keeping higher carbon concentration and higher pH can improve CO2 absorption ratio, which will optimize the biofixation of CO2 by microalgae furthermore.

The diversity, origin, and evolutionary analysis of geosmin synthase gene in cyanobacteria.

The sesquiterpene geosmin, mainly originating from cyanobacteria, is considered one of the problematic odor compounds responsible for unpleasant-tasting and -smelling water episodes in freshwater supplies. The biochemistry and genetics of geosmin synthesis in cyanobacteria is well-elucidated and the geosmin synthase gene (geo) has been cloned and characterized in recent years. However, understanding the diversity, origin, and evolution of geo has been hindered by the limited availability of geo sequences to date. On the basis of the cloned geo sequences from16 filamentous geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales, the diversity and evolution of geo in cyanobacteria was systematically analyzed in this study. Homologous alignment revealed that geo is highly conserved among the examined cyanobacterial species, with DNA sequence identities >0.72. Phylogenetic reconstruction and codon bias analysis based on geo suggest that cyanobacterial geo form a monophyletic branch with a common origin and ancestor for cyanobacteria, actinomycetes, and myxobacteria. The global ratio of nonsynonymous/synonymous nucleotide substitutions (dN/dS) was 0.125, which is substantially <1 and indicates strong purifying selection in the evolution of cyanobacterial geo. To add to further interest, horizontal gene transfer of cyanobacterial geo in evolutionary history was confirmed by the discovery of an incongruent coevolutionary relationship between geo and housekeeping genes 16S rDNA and rpoC. The present study enhances the fundamental understanding of cyanobacterial geo in diversity and evolution, and sheds light on the development of molecular assays for detection and molecular ecology research of geosmin-producing cyanobacteria.

Integrated lipid production, CO2 fixation, and removal of SO2 and NO from simulated flue gas by oleaginous Chlorella pyrenoidosa.

CO2, SO2, and NO are the main components of flue gas and can cause serious environmental issues. Utilization of these compounds in oleaginous microalgae cultivation not only could reduce air pollution but could also produce feedstock for biodiesel production. However, the continuous input of SO2 and NO inhibits microalgal growth. In this study, the toxicity of simulated flue gas (15% CO2, 0.03% SO2, and 0.03% NO, balanced with N2) was reduced through automatic pH feedback control. Integrated lipid production and CO2 fixation with the removal of SO2 and NO was achieved. Using this technique, a lipid content of 38.0% DW was achieved in Chlorella pyrenoidosa XQ-20044. The lipid composition and fatty acid profile indicated that lipid production by C. pyrenoidosa XQ-20044 cultured with flue gas is suitable as a biodiesel feedstock; 81.2% of the total lipids were neutral lipids and 99.5% of the total fatty acids were C16 and C18. The ratio of saturated fatty acids to monounsaturated fatty acids in the microalgal lipid content was 74.5%. In addition, CO2, SO2, and NO from the simulated flue gas were fixed and converted to biomass and lipids with a removal efficiency of 95.9%, 100%, and 84.2%, respectively. Furthermore, the utilization efficiencies of CO2, SO2, and NO were equal to or very close to their removal efficiencies. These results provide a novel strategy for combining biodiesel production with biofixation of flue gas.

Sequential phototrophic-mixotrophic cultivation of oleaginous microalga Graesiella sp. WBG-1 in a 1000 m2 open raceway pond.

BACKGROUND: Microalgae are an important feedstock in industries. Currently, efforts are being made in the non-phototrophic cultivation of microalgae for biomass production. Studies have shown that mixotrophy is a more efficient process for producing algal biomass in comparison to phototrophic and heterotrophic cultures. However, cultivation of microalgae in pilot-scale open ponds in the presence of organic carbon substrates has not yet been developed. The problems are heterotrophic bacterial contamination and inefficient conversion of organic carbon. RESULTS: Laboratory investigation was combined with outdoor cultivation to find a culture condition that favors the growth of alga, but inhibits bacteria. A window period for mixotrophic cultivation of the alga Graesiella sp. WBG-1 was identified. Using this period, a new sequential phototrophic-mixotrophic cultivation (SPMC) method that enhances algal biomass productivity and limits bacteria contamination at the same time was established for microalgae cultivation in open raceway ponds. Graesiella sp. WBG-1 maximally produced 12.5 g biomass and 4.1 g lipids m-2 day-1 in SPMC in a 1000 m2 raceway pond, which was an over 50% increase compared to phototrophic cultivation. The bacterial number in SPMC (2.97 × 105 CFU ml-1) is comparable to that of the phototrophic cultivations. CONCLUSIONS: SPMC is an effective and feasible method to cultivate lipid-rich microalgae in open raceway ponds. Successful scale-up of SPMC in a commercial raceway pond (1000 m2 culture area) was demonstrated for the first time. This method is attractive for global producers of not only lipid-rich microalgae biomass, but also astaxanthin and ß-carotene.

Maximizing CO2 biofixation and lipid productivity of oleaginous microalga Graesiella sp. WBG-1 via CO2-regulated pH in indoor and outdoor open reactors.

Carbon dioxide (CO2) and pH are two interdependent factors that greatly impact the growth and lipid accumulation of microalgae. However, the effects of these two factors are usually studied separately. The use of exogenous CO2, such as flue gas derived, to regulate pH in the large-scale cultivation of microalgae provides an ideal means for combining CO2 biofixation and biodiesel production. In this study, the CO2 biofixation and lipid production of oleaginous microalga Graesiella sp. WBG-1 was explored for four pH levels regulated by exogenous 15% CO2 (flue gas concentration) in 10L circular culture ponds and 5m2 open raceway reactors. Results revealed that pH8.0-9.0 was the optimum pH for CO2 fixation and lipid production, attaining the highest CO2 fixation rates of 0.26gL-1day-1 and 18.9gm-2day-1, respectively, lipid contents of 46.28% and 32.38%, and lipid productivities of 64.8mgL-1day-1 and 3.14gm-2day-1. A positive correlation between CO2 utilization efficiency and pH in open reactors was also suggested in this research, and thus provides direction for screening of CO2 fixation by microalgae. The present study provides an excellent strategy for coupling CO2 fixation and lipid production via microalgae in large-scale cultivation.

Effective cultivation of microalgae for biofuel production: a pilot-scale evaluation of a novel oleaginous microalga Graesiella sp. WBG-1.

BACKGROUND: Commercial production of microalgal biodiesel is not yet economically viable, largely because of low storage lipid yield in microalgae mass cultivation. Selection of lipid-rich microalgae, thus, becomes one of the key research topics for microalgal biodiesel production. However, the laboratory screening protocols alone cannot predict the ability of the strains to dominate and perform in outdoor ponds. Comprehensive assessment of microalgae species should be performed not only under the laboratory conditions, but also in the fields. RESULTS: Laboratory investigations using a bubbled column photobioreactor indicated the microalga Graesiella sp. WBG-1 to be the most productive species among the 63 Chlorophyta strains. In a 10 L reactor, mimicking the industrial circular pond, Graesiella sp. WBG-1 produced 12.03 g biomass m(-2) day(-1) and 5.44 g lipids (45.23 % DW) m(-2) day(-1) under 15 mol m(-2) day(-1) artificial light irradiations. The lipid content decreased to ~34 % DW when the microalga was cultured in 30 L tank PBR under natural solar irradiations, but the decline of lipid content with scaling up was the minimum among the tested strains. Based on these results, the microalga was further tested for its lipid production and culture competitiveness using a pilot-scale raceway pond (200 m(2) illuminated area, culture volume 40,000 L). Consequently, Graesiella sp. WBG-1 maintained a high lipid content (33.4 % DW), of which ~90 % was storage TAGs. Results from the outdoor experiments indicated the nice adaptability of the Graesiella sp. WBG-1 to strong and fluctuating natural solar irradiance and temperature, and also demonstrated several other features, such as large cell size (easy for harvest and resistant to swallow by protozoa) and tolerance to high culture pH (helpful to CO2 fixation). CONCLUSIONS: Graesiella sp. WBG-1 was a promising strain capable of accumulating large amount of storage lipid under nature solar irradiance and temperature. The high lipid content of 33.4 % DW was achieved for the first time in pilot-scale raceway pond. The results also provide evidence for the feasibility of using low-cost raceway pond for autotrophic cultivation of microalgae for biodiesel production.

The Effects of Physicochemical Factors and Cell Density on Nitrite Transformation in a Lipid-Rich Chlorella.

To understand the effects of physicochemical factors on nitrite transformation by microalgae, a lipid-rich Chlorella with high nitrite tolerance was cultured with 8 mmol/l sodium nitrite as sole nitrogen source under different conditions. The results showed that nitrite transformation was mainly dependent on the metabolic activities of algal cells rather than oxidation of nitrite by dissolved oxygen. Light intensity, temperature, pH, NaHCO3 concentrations, and initial cell densities had significant effects on the rate of nitrite transformation. Single-factor experiments revealed that the optimum conditions for nitrite transformation were light intensity: 300 µmol/m(2); temperature: 30°C; pH: 7-8; NaHCO3 concentration: 2.0 g/l; and initial cell density: 0.15 g/l; and the highest nitrite transformation rate of 1.36 mmol/l/d was achieved. There was a positive correlation between nitrite transformation rate and the growth of Chlorella. The relationship between nitrite transformation rate (mg/l/d) and biomass productivity (g/l/d) could be described by the regression equation y = 61.3x (R(2) = 0.9665), meaning that 61.3 mg N element was assimilated by 1.0 g dry biomass on average, which indicated that the nitrite transformation is a process of consuming nitrite as nitrogen source by Chlorella. The results demonstrated that the Chlorella suspension was able to assimilate nitrite efficiently, which implied the feasibility of using flue gas for mass production of Chlorella without preliminary removal of NOX.

Isolation and characterization of a new reported cyanobacterium Leptolyngbya bijugata coproducing odorous geosmin and 2-methylisoborneol.

The earthy-musty compounds geosmin and 2-methylisoborneol (MIB) produced by cyanobacteria are considered as the main biological causes of off-flavor events, especially in aquatic ecosystems. More than 50 filamentous cyanobacteria species have been documented as geosmin or MIB producers; however, little is known about the species coproducing these two metabolites. In this study, an epiphytic sample was collected from a river in Hubei, China. Three isolated strains (A2, B2, and B4) producing earthy odors were successfully isolated and identified as the cyanobacterium Leptolyngbya bijugata Anagnostidis et Komárek 1988 based on morphology and 16S rDNA sequences. Gas chromatography analysis confirmed that the isolated L. bijugata strains were geosmin and MIB coproducers, with accumulation ranging from 13.6 to 22.4 and 12.3 to 57.5 µg L(-1), respectively. The partial fragments of geosmin and MIB synthesis genes in the L. bijugata strains were cloned and sequenced. Further sequences and phylogenetic analysis indicated the high conservation and a common origin of these genes in cyanobacteria. This study is the first to report and characterize the coproduction of geosmin and MIB by L. bijugata, representing a new source for potential risk of off-flavor events.

Enhanced lipid production in Chlorella pyrenoidosa by continuous culture.

Usually microalgae growth and lipid accumulation do not run in parallel throughout cultivation, which necessarily lowers overall lipid productivity. However, we show through batch and feed-batch studies of Chlorella pyrenoidosa XQ-20044 that by varying the nitrate concentration, conditions which produce fairly high lipid content could be achieved without sacrificing algal growth. Simultaneous microalgae growth and lipid production was achieved in continuous chemostat culture when the specific nitrate input rate was in the range of 0.78-4.56mmolg(-1)d(-1). Moreover, the maximum lipid productivity (144.93mgL(-1)d(-1)) in the continuous culture was significantly higher than in batch culture (96.28mgL(-1)d(-1)), thus indicating the feasibility and great advantage of one-step production of microalgal lipids.

Growth rate and biomass productivity of chlorella as affected by culture depth and cell density in an open circular photobioreactor.

The effects of culture depth (2-10 cm) and cell density on the growth rate and biomass productivity of Chlorella sp. XQ-200419 were investigated through the use of a selfdesigned open circular pond photobioreactor-imitation system. With increases in culture depths from 2 to 10 cm, the growth rate decreased significantly from 1.08 /d to 0.39 /d. However, the biomass productivity only increased slightly from 8.41 to 11.22 g/m2/d. The biomass productivity (11.08 g/m2/d) achieved in 4 cm culture with an initial OD540 of 0.95 was similar to that achieved in 10 cm culture with an initial OD540 of 0.5. In addition, the duration of maximal areal productivity at a 4 cm depth was prolonged from 1 to 4 days, a finding that was also similar to that of the culture at a 10 cm depth. In both cases, the initial areal biomass densities were identical. Based on these results and previous studies, it can be concluded that the influence of culture depth and cell density on areal biomass productivity is actually due to different areal biomass densities. Under suitable conditions, there are a range of optimal biomass densities, and areal biomass productivity reaches its maximum when the biomass density is within these optimal ranges. Otherwise, biomass productivity will decrease. Therefore, a key factor for high biomass productivity is to maintain an optimal biomass density.

Isolation and characterization of a mesophilic Arthrospira maxima strain capable of producing docosahexaenoic acid.

A strain of the cyanobacterium Arthrospira was isolated from Lake Chahannaoer in northern China and was characterized according to microscopic morphology, photosynthetic oxygen-evolving activity, growth rate, and nutritional profile. Compared with thermophilic Arthrospira species occurring naturally in tropical and subtropical lakes, this isolate is mesophilic and grows optimally at ~20 degreesC. The total protein, fatty acid, phycocyanin, carotenoid, and chlorophyll a contents were 67.6, 6.1, 4.32, 0.29, and 0.76 grams per 100 grams of dry weight, respectively. The strain is rich in polyunsaturated fatty acids (PUFAs). An essential omega-3 fatty acid, docosahexaenoic acid (DHA), was detected, and gamma-linolenic acid (GLA) and DHA accounted for 28.3% of the total fatty acid content. These features of this newly isolated strain make it potentially useful in commercial mass culture in local areas or as a biofuel feedstock. It is also an alternative resource for studying the metabolic PUFA pathways and mechanisms of cold stress tolerance in cyanobacteria.

The synthesis of astaxanthin esters, independent of the formation of cysts, highly correlates with the synthesis of fatty acids in Haematococcus pluvialis.

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.

Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

After first being analyzed by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters in Haematococcus pluvialis were identified by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-(APCI)MS). Identification of each compound was based on the characteristic fragment ions of the positive ion mode, negative ion mode, and MS(2). Astaxanthin esters were identified based on the loss of one or two fatty acids. In a positive ion mode, astaxanthin monoesters had characteristic fragment ions at m/z 597 [M+H-fatty acid](+) and m/z 579 and 561 that resulted from a continuous loss of water. The relative intensity of m/z 579 in MS(2) amounted to more than 80% of that of the molecular ion. In astaxanthin diesters, the intensity of m/z 561 occasionally was equal to that of m/z 579, but in general the former, amounting to 50 to 60% or more of the molecular ion, was stronger than the latter, which decreased to 20 to 30% of the molecular ion. In addition, a set of compounds with maximum absorbance at 400 nm, detected by high-performance liquid chromatography-diode array detector (HPLC-DAD), had strong characteristic fragment ions at m/z 871 and 593 in the positive ion mode MS(2). They were presumed to be linolenic acid or an isomer of omega-6-gamma-linolenic acid esters of astacin.

[Laboratory culture of Tetraselmis strains isolated from the coasts of China].

Tetraselmis is an important alga commonly used as live food in aquaculture. This paper reported the laboratory culture of its strains collected from the coasts of China, covering Guangdong, Zhejiang and Shandong Provinces. The separated strains of Tetraselmis for mono-species culture were isolated with capillary method, and their axenic cultures were obtained by inoculating them in plates with solid Marine III medium (M III). Modified Guillard & Ryther medium (MGM), M III, and modified Marine III medium (M III M) were utilized for the liquid cultivation of Tetraselmis in 500 mL flasks. It was displayed that the average growth of Tetraselmis cultured in M III M was the fastest at pH 7.0-8.0, and vitamin B12 addition to M III M slightly increased the relative growth rate of Tetraselmis. Therefore, the modified Marine III medium was effective to use as a large-scale culture medium for Tetraselmis in aquaculture.