Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

镧系元素对海水小球藻的毒性效应

用评价化学品对藻类毒性的标准实验方法,研究了12种镧系元素对小球藻生长的抑制情况。结果表明,12种镧系元素抑制小球藻生长的96h半效应浓度(96h EC50 )均为2 9 0 0±0 5 0 μmol·L-1。对各剂量反应方程进行X2 检验,结果表明,符合精度要求,计算出的96h EC50 真实可靠。镧系元素对海水小球藻的生物毒性是相同的,此结果对探明镧系元素对藻类的生态毒理效应具有重要意义。     

Phospholipid Metabolism in an Industry Microalga Chlorella sorokiniana: The Impact of Inoculum Sizes

Chlorella sorokiniana is an important industry microalga potential for biofuel production. Inoculum size is one of the important factors in algal large-scale culture, and has great effects on the growth, lipid accumulation and metabolism of microalgae. As the first barrier of cell contents, membrane plays a vital role in algal inoculum-related metabolism. The knowledge of phospholipids, the main membrane component and high accumulation of phospholipids as the major content of total lipids mass in some microalgae, is necessary to understand the role of membrane in cell growth and metabolism under different inoculum density. Profiling of C. sorokiniana phospholipids with LC-MS led to the identification of 119 phospholipid species. To discover the phospholipid molecules most related to change of inoculum sizes, Partial Least Squares Discriminant Analysis (PLS-DA) was employed and the results revealed that inoculum sizes significantly affected phospholipid profiling. Phosphatidylglycerol (PG), phosphatidyl- ethanolamine (PE) and several phosphatidylcholine (PC) species might play an important role under our experimental conditions. Further analysis of these biomarkers indicated that cell membrane status of C. sorokiniana might play an important role in the adaption to the inoculum sizes. And the culture with inoculum size of 1×10⁶ cells mL⁻¹ presented the best membrane status with the highest content of PC and PG, and the lowest content of PE. We discovered that the inoculum size of 1×10⁶ cells mL⁻¹ might provide the best growth condition for C. sorokiniana. Also we proposed that PG, PE and several PC may play an important role in inoculum-related metabolism in C. sorokiniana, which may work through thylakoid membrane and photosynthetic pathway. Thus this study would provide more potential targets for metabolic engineering to improve biofuel production and productivity in microalgae.     

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K_m values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.     

Nitrogen Metabolism Genes from Temperate Marine Sediments

In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO₃ ^? was present in the top 5 cm below the sediment-water interface at both sites. NH₄ ⁺ increased with depth below 5 cm where it overlapped with the NO₃ ^? zone. Steady-state modelling of NO₃ ^? and NH₄ ⁺ porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dissimilatory NO₃ ^? reduction to NH₄ ⁺ (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO₃ ^? reduction to NO₂ ^? or NH₄ ⁺ as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

Metabolism of Urea by Chlorella vulgaris

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.     

Isolation and Characterization of Antifreeze Proteins from the Antarctic Marine Microalga Pyramimonas gelidicola

Antifreeze proteins (AFPs) play an important role in the psychrophilic adaptation of polar organisms. AFPs encoded by an Antarctic chlorophyte, identified as Pyramimonas gelidicola, were isolated and characterized. Two AFP isoforms were found from cDNAs and their deduced molecular weights were estimated to be 26.4 kDa (Pg-1-AFP) and 27.1 kDa (Pg-2-AFP). Both AFP cDNAs were cloned and expressed in Escherichia coli. The purified recombinant Pg-1-rAFP and Pg-2-rAFP both showed antifreeze activity based on the measurement of thermal hysteresis (TH) and morphological changes to single ice crystals. Pg-1-rAFP shaped ice crystals into a snowflake pattern with a TH value of 0.6?±?0.02 °C at ~15 mg/ml. Single ice crystals in Pg-2-rAFP showed a dendritic morphology with a TH value of 0.25?±?0.02 °C at the same protein concentration. Based on in silico protein structure predictions, the three-dimensional structures of P. gelidicola AFPs match those of their homologs found in fungi and bacteria. They fold as a right-handed β-helix flanked by an α-helix. Unlike the hyperactive insect AFPs, the proposed ice-binding site on one of the flat β-helical surfaces is neither regular nor well-conserved. This might be a characteristic of AFPs used for freeze tolerance as opposed to freeze avoidance. A role for P. gelidicola AFPs in freeze tolerance is also consistent with their relatively low TH values.     

Patterns of Post-Glacial Genetic Differentiation in Marginal Populations of a Marine Microalga

This study investigates the genetic structure of an eukaryotic microorganism, the toxic dinoflagellate Alexandrium ostenfeldii, from the Baltic Sea, a geologically young and ecologically marginal brackish water estuary which is predicted to support evolution of distinct, genetically impoverished lineages of marine macroorganisms. Analyses of the internal transcribed spacer (ITS) sequences and Amplified Fragment Length Polymorphism (AFLP) of 84 A. ostenfeldii isolates from five different Baltic locations and multiple external sites revealed that Baltic A. ostenfeldii is phylogenetically differentiated from other lineages of the species and micro-geographically fragmented within the Baltic Sea. Significant genetic differentiation (F _ST) between northern and southern locations was correlated to geographical distance. However, instead of discrete genetic units or continuous genetic differentiation, the analysis of population structure suggests a complex and partially hierarchic pattern of genetic differentiation. The observed pattern suggests that initial colonization was followed by local differentiation and varying degrees of dispersal, most likely depending on local habitat conditions and prevailing current systems separating the Baltic Sea populations. Local subpopulations generally exhibited low levels of overall gene diversity. Association analysis suggests predominately asexual reproduction most likely accompanied by frequency shifts of clonal lineages during planktonic growth. Our results indicate that the general pattern of genetic differentiation and reduced genetic diversity of Baltic populations found in large organisms also applies to microscopic eukaryotic organisms.     

13C-Tracer and Gas Chromatography-Mass Spectrometry Analyses Reveal Metabolic Flux Distribution in the Oleaginous Microalga Chlorella protothecoides

The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-¹³C]/[1-¹³C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts.Due to the energy shortage, rising petroleum prices, and the environmental impact of fossil energy-based industries, oleaginous microalgae are receiving increasing attention as a potential feedstock for providing a large source of renewable fuel materials. Biofuel from microalgae (the third-generation biofuel) offers obvious environmental benefits because the process can be coupled with photosynthetic carbon dioxide mitigation (Wang et al., 2008; Brune et al., 2009). Besides this, other eco-friendly behaviors of microalgae also garner interest, including that some algal species can remove nitrogen oxide from combustion gases, grow well in brackish habitats, use far less water than traditional oilseed crops, and double their numbers within 1 d, resulting in increased biomass yield (Li et al., 2008).The economical and environmental benefits of microalgal biofuel prompted research and development of algal strains that synthesize a high proportion of oil. The green alga Chlorella protothecoides is one of the best oil-producing species ever reported (Rosenberg et al., 2008). Under heterotrophic culture conditions, it rapidly transforms carbohydrates into triacylglycerols (more than 50% of dry cell weight), which can be further transesterified with alcohol (Miao and Wu, 2006; Xu et al., 2006; Xiong et al., 2008). These processes open a promising and highly efficient pathway for biodiesel production. A crucial point affecting biodiesel refinery is the oil productivity of heterotrophic Chlorella, which primarily relies on carbon flow from sugar to oil. Since the backbone of biomass components and by-products (e.g. proteins, CO₂) besides triacylglycerols are also derived from carbon substrate, the final yield of oil is determined by the intracellular distribution of carbon flux. In our previous work (Miao and Wu, 2006), carbon flux targeting into lipid synthesis was severely affected by the nutrient environment of the medium. Oil accumulation tends to occur under carbon-sufficient but nitrogen-limited conditions (Rosenberg et al., 2008), reflecting a unique way in which metabolic networks of Chlorella respond to environmental perturbations. However, until now, in-depth knowledge of the metabolic network in green alga was limited by the lack of quantitative data. Thus, it is urgently required to gain metabolic information on microalgae to better understand the intracellular distribution of carbon fluxes in response to environmental stimuli.On the basis of ¹³C-labeling experiments, metabolic flux analysis (MFA) emerged as an integrated experimental/computational tool to identify the biochemical network of active reactions and to provide quantitative insight into the in vivo distribution of molecular fluxes throughout central carbon metabolism (Zamboni et al., 2009). The general principle of this cutting-edge methodology is based on ¹³C-tracer study, which can distinguish fluxes through different pathways when these fluxes lead to different positional isotopic enrichments. These labeling patterns are imprinted in metabolic intermediates (e.g. protein-bound amino acids) and can be analyzed by gas chromatography-mass spectrometry (GC-MS) or NMR spectroscopy. Fluxomic information then can be quantified from the isotope data by mathematical modeling. For instance, an alternative approach, named metabolic flux ratio analysis (Fischer and Sauer, 2003), is to utilize algebraic equations for determining strictly local ratios of converging fluxes. Absolute intracellular fluxes may be further assessed by implementing such flux partitioning ratios to a linear equation system, which results from material balances (e.g. ¹³C constraint flux analysis; Fischer et al., 2004). Following these procedures, ¹³C MFA has the ability to resolve parallel, cyclic, and reversible fluxes, making it a powerful technique not only for quantifying metabolic fluxes but also for identifying novel or unexpected metabolic pathways. In recent years, the successful application of the ¹³C-flux method for determining the in vivo reaction velocities in model microorganisms, such as Escherichia coli (Yang et al., 2003), Bacillus subtilis (Fischer and Sauer, 2005), Corynebacterium glutamicum (Hoon Yang et al., 2006), and Saccharomyces cerevisiae (Blank et al., 2005), has been widely reported. Moreover, the ¹³C-flux method also demonstrated its value in tracking metabolic profiles in plant and animal cells. For example, recent research on heterotrophic cell suspension cultures of Arabidopsis (Arabidopsis thaliana) highlighted the stability of the flux distribution under different oxygenation conditions (Williams et al., 2008), while work on breast tumor cells revealed widespread changes to central metabolism upon cellular transformation (Yang et al., 2008). Nevertheless, to our knowledge, this approach has yet to be applied to eukaryotic alga grown heterotrophically.In recent decades, heterotrophic fermentation of photosynthetic microorganisms was raised as an important strategy to improve the efficiency and reduce the cost of alga-based biorefinery. Particularly, the heterotrophic growth of Chlorella is of wide concern for commercial production of high-value carotenoid (Sansawa and Endo, 2004), lutein (Shi et al., 2002), astaxanthin (Del Campo et al., 2004), and even biofuels (Xiong et al., 2008). Nevertheless, compared with other photosynthetic organisms, Chlorella grown on organic substrates is less understood due to the smaller data set of accurate genomic and biochemical information than is typically available for model plants. Here, we demonstrate that by using well-designed ¹³C-tracer experiments and highly sensitive isotopomer analysis, quantitative metabolic knowledge in not fully characterized species can be obtained. We adopted GC-MS to analyze the labeling patterns of the amino acids in biomass hydrolysates of Chlorella grown in a chemically defined medium with different carbon-nitrogen (C/N) ratios. Two-dimensional ¹H-¹³C NMR spectroscopy was further utilized to confirm flux ratios in key nodes of the Chlorella metabolic network. By integrating these labeling measurement data with metabolite balancing, the intracellular flux distributions in Chlorella were thus quantitated. This article is, to our knowledge, the first ¹³C MFA study for identifying and quantifying the intracellular metabolic fluxes in oleaginous alga.     

Growth-Promoting Effect of Alginate Oligosaccharides on a Unicellular Marine Microalga,Nannochloropsis oculata

Effects of an alginate oligosaccharide mixture (AOM) on Nannochloropsis oculata, a unicellular marine microalga, were investigated. The growth of N. oculata was significantly promoted by AOM in a concentration-dependent manner. The maximum effect was attained at 20 mg/ml, at which the growth rate of the alga became nearly 5 times higher than that of control without AOM. The growth-promoting effect of AOM decreased slightly at 40 mg/ml. Furthermore, the algicidal effect of Cu²⁺ was profoundly alleviated by the addition of AOM. These results suggest that AOM is useful for promoting and/or improving the growth of N. oculata.     

Nitrogen Metabolism of the Human Brain

Cerebral nitrogen metabolism was studied in 29 healthy nonobese volunteers by means of a catheterization technique. Arterial levels and arterial-jugular venous (A-JV) concentration differences for amino acids, urea, ammonia, 5-oxoproline, glucose, and oxygen were measured in the basal, postabsorptive state and during an intravenous infusion of a commercial amino acid solution. In the basal state positive A-JV differences, indicating a net brain uptake, were noted for 12 of 22 amino acids as well as for ammonia. There was no significant net exchange for urea or for 5-oxoproline. During amino acid infusion, resulting in a 150-300% rise in arterial amino acid levels, the brain uptake of isoleucine, leucine, and tyrosine increased significantly, and a similar tendency was seen for most other amino acids. The infusion was accompanied by a 100% rise in arterial ammonia levels and a 10% increase in urea concentration. For ammonia the small positive A-JV difference in the basal state became markedly greater during amino acid infusion, whereas no significant alteration was noted for urea exchange across the brain. The A-JV differences for glucose and oxygen were positive in the basal state and unchanged during the infusion. The present findings demonstrate that in the basal state (a) there is a significant net brain uptake of most amino acids; (b) no single amino acid, urea, or 5-oxoproline is released from the brain; and (c) ammonia uptake occurs both in this state and during an amino acid infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of Fatty Acid Hydroperoxides by Chlorella pyrenoidosa

The green alga Chlorella pyrenoidosa was examined for its ability to metabolize 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. The study showed that Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella. In vitro enzyme studies also revealed the presence of hydroperoxide lyase activity that cleaved 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acid into two products, 13-oxo-cis-9,trans-11-tridecadienoic acid and pentane (from linoleic acid) or pentene (from linolenic acid). The lyase was heat-labile, insensitive to 50 millimolar KCN, and had an approximate molecular weight of 48,000 as estimated by gel filtration. Two other products, 13-hydroxy-cis-9,trans-11,cis-15-octadecatrienoic acid and 12, 13-trans-epoxy-9-oxo-trans-10,cis-15-octadecadienoic acid, were also observed. Because these compounds are also products of nonenzymic, Fe(II)-catalyzed hydroperoxide decomposition reactions, their presence suggested that the observed lyase activity may occur via a homolytic decomposition mechanism.     

Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 μg of fGH protein expression per one liter culture containing 1 × 10⁸ cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding. Received March 26, 2001; accepted July 10, 2001.