Correlation between lipid and carotenoid synthesis in torularhodin-producing <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The oleaginous red yeast Rhodotorula glutinis produces carotenoid pigments, especially torularhodin and β-carotene, in significant amounts. We have analyzed in detail carotenoid and lipid biosynthesis in a torularhodin-producing strain of R. glutinis cultivated at different carbon:nitrogen (C/N) ratios (20:1, 50:1, 70:1, and 100:1). When the strain was cultivated in media with low C/N ratios (20:1 and 50:1), glucose was completely utilized and carotenoid formation was stimulated. Maximum pigment production reached 12.9 mg/L of medium and 2.3 mg/g of biomass at the C/N ratio of 20:1. It was noted that β-carotene synthesis was prominent when glucose was present in the medium. However, glucose exhaustion in the media at C/N ratios of 20:1 and 50:1 was closely accompanied by the predominant formation of torularhodin. The growth of R. glutinis in media with C/N ratios of 70:1 and 100:1 favored lipid accumulation in the cells but carotenoid biosynthesis was reduced. In addition, glucose consumption was linked to a rapid decrease in oleic acid levels in the total intracellular lipids. The kinetic analysis clearly indicated a correlation between oleic acid levels in total lipids and torularhodin accumulation in the cells. The results may suggest that acetyl-CoA formed from oleic acid degradation is metabolized through the mevalonate/isoprenoid/carotenoid pathways directly to torularhodin.     

Effect of different C/N ratios on carotenoid and lipid production by Rhodotorula glutinis

Due to the increasing demand for sustainable biofuels, microbial oils as feedstock for the transesterification into biodiesel have gained scientific and commercial interest. Also, microbial carotenoids have a considerable market potential as natural colorants. The carbon to nitrogen (C/N) ratio of the respective cultivation media is one of the most important parameters that influence the production of microbial lipids and carotenoids. Thus, in the present experiment, the influence of different C/N ratios, initial glucose loadings, and ammonium concentrations of the cultivation medium on microbial cell growth and lipid and carotenoid production by the oleaginous red yeast Rhodotorula glutinis has been assessed. As a general trend, both lipid and carotenoid production increased at high C/N ratios. It was shown that not only the final C/N ratio but also the respectively applied initial carbon and nitrogen contents influenced the observed parameters. The lipid yield was not affected by different ammonium contents, while the carotenoid production significantly decreased both at low and high levels of ammonium supply. A glucose-based increase from C/N 70 to 120 did not lead to an increased lipid production, while carotenoid synthesis was positively affected. Generally, it can be asserted that lipid and carotenoid synthesis are stimulated at higher C/N ratios.     

Seasonal changes in lipid content and composition of the polychaete Nereis (Hediste) diversicolor

The lipid content and composition of Nereis (Hediste) diversicolor O. F. Müller (Annelida, Polychaeta, Nereidae) a mud-dwelling, intertidal errant polychaete in the Tagus estuary (Portugal), were examined on the monthly basis by lipid extraction, TLC and capillary GC. In this estuary, N. diversicolor is by far the dominant species among polychaeta and the main food item in the natural diet of several flatfishes. The biochemical elucidation of its lipid structure and distribution throughout the year, described in this study, provides information not only about the physiological role of lipids in the animal under consideration but also about dietary fatty acid requirements of some flatfishes in the wild and under laboratory conditions.The total lipid content varied between a maximum of 19.3% lyophilized dry weight in February (4.4% fresh weight) and a minimum of 6.6% in August (1.9% fresh weight). The major lipid classes were triacylglycerol, phospholipid, free sterol, free fatty acid, sterol ester/wax ester and alkyldiacylglycerol.The fatty acid composition was rather unsaturated with a 1:2 mean ratio of n-3: n-6. The major fatty acids were C160:0, C18:1n-9, C18:2n-6, and C20:5n-3; there were smaller amounts of C180:0, C18:1n-11, C18:1n-7, C18:3n-3, C20:1, C20:2n-6, C20:4n-6, C22:2, C22:5n-3, and many other fatty acids were detected at trace levels. The unsaturation ranged from 36.9 mg/g dry weight in summer to 107.4 mg/g in winter. An accumulation of fatty acids from plant origin was evident, in particular linoleic acid (C18:2n-6), which was quantitatively one of the major fatty acids throughout the year.     

Effect of C/N ratio and aeration on the fatty acid composition of heterotrophicChlorella sorokiniana

The effect of the carbon to nitrogen (C/N) ratio of the medium and the aeration rate on the lipid content and fatty acid composition ofChlorella sorokiniana was investigated using heterotrophic, batch culture. Both parameters had a significant effect. A C/N ratio of approximately 20, was found to indicate a change from carbon to nitrogen limitation forC. sorokiniana. Cell lipid content was at a minimum at this value and increased at both higher and lower C/N values. Low C/N ratios favoured a high proportion of trienoic fatty acids at the expense of monoenoic acids. Aeration enhanced cell growth, fatty acid yield and the synthesis of unsaturated dienoic and trienoic fatty acids, but reduced cell lipid content. The results demonstrate that the fatty acid composition and lipid content of heterotrophically-grown microalgae can be favourably manipulated by varying culture conditions.     

Composition of the lipids of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> and their origin from its host <Emphasis Type="Italic">Ignicoccus</Emphasis> sp. strain KIN4/I

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent ¹³C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.     

Efficient concomitant production of lipids and carotenoids by oleaginous red yeast <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> cultured in palm oil mill effluent and application of lipids for biodiesel production

Rhodotorula glutinis TISTR 5159 is oleaginous red yeast that accumulates both lipids and carotenoids. It was cultured in palm oil mill effluent (POME) with only the addition of ammonium sulfate and Tween 20 as a suitable nitrogen source and surfactant, respectively. Response surface methodology (RSM) was applied to optimize initial chemical oxygen demand (COD) in POME, C/N ratio, and Tween 20 concentration for concomitant production of lipids and carotenoids. Among three investigated factors, C/N ratio contributed a significant effect upon lipid and carotenoids production. Analysis of response surface plots revealed that the optimum C/N ratio for the biomass was 140, while that for lipid content and carotenoids were higher at 180 and 170, respectively. The high level of the nitrogen source (with a low C/N ratio) enhanced the biomass, making the accumulation of lipids and carotenoids less preferable. Hence, the two-stage process was attempted as an optimal way for cell growth in the first stage and product accumulation in the second stage. The lipid yield and carotenoid production obtained in the two-stage process were higher than those in the one-stage process. In the semi-continuous fermentation, R. glutinis TISTR 5159 accumulated high lipid content and produced a considerably high concentration of carotenoids during long-term cultivation. Additionally, efficient COD removal by R. glutinis TISTR 5159 was observed. The biodiesel produced from yeast lipids was composed mainly of oleic and palmitic acids, similar to those from plant oil.     

Ontogenetic changes in biochemical composition during larval and early postlarval development of Lepidophthalmus louisianensis, a ghost shrimp with abbreviated development

Changes in growth and biochemical composition during the transition from egg through zoea to decapodid in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935), were documented in terms of dry weight, lipid classes, fatty acid composition, and carbon to nitrogen (C:N) ratios. Larvae of the ghost shrimp were mass-reared in the laboratory (28°C; 20‰ S) from hatching to the decapodid stage. Iatroscan lipid class analysis revealed that major lipid classes in recently produced eggs were phospholipids (80.8±1.3%) and triglycerides (16.0±1.1%), which decreased during the incubation period. Polar lipids (zoea I: 77.4±1.7%; zoea II: 77.5±2.1%; decapodid: 80.0±1.7%) and neutral lipids, of which free fatty acids (zoea I: 10.5±2.7%; zoea II: 13.1±5.2%; decapodid: 7.8±2.1%) were dominant, represented the major lipid classes in the zoeal and decapodid stages. Triglycerides were present in small amounts. The predominant fatty acids of L. louisianensis eggs, zoeae and decapodids were palmitic (16:0), stearic (18:0), eicosapentaenoic (20:5ω3), oleic (18:1ω9), and arachidonic (20:4ω6). Elemental composition of eggs, larvae, and the decapodid stage revealed conspicuous changes in the C:N ratio, with N being relatively stable during larval development but C decreasing during the decapodid stage. These data suggest independence of newly hatched L. louisianensis on external energy resources. This combined with the ability to incorporate saturated fatty acids into polar lipids provides a selective advantage for fast development of new tissue and growth, characteristic of decapod crustacean larvae with lecithotrophic development.     

Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi

Effects of carbon concentration and carbon to nitrogen (C:N) ratio on six biocontrol fungal strains are reported in this paper. All fungal strains had extensive growth on the media supplemented with 6–12 g l⁻¹ carbon and C:N ratios from 10:1 to 80:1, and differed in nutrient requirements for sporulation. Except for the two strains of Paecilomyces lilacinus, all selected fungi attained the highest spore yields at a C:N ratio of 160:1 when the carbon concentration was 12 g l⁻¹ for Metarhizium anisopliae SQZ-1-21, 6 g l⁻¹ for M. anisopliae RS-4-1 and Trichoderma viride TV-1, and 8 g l⁻¹ for Lecanicillium lecanii CA-1-G. The optimal conditions for P. lilacinus sporulation were 8 g l⁻¹ carbon with a C:N ratio of 10:1 for M-14 and 12 g l⁻¹ carbon with a C:N ratio of 20:1 for IPC-P, respectively. The results indicated that the influence of carbon concentration and C:N ratio on fungal growth and sporulation is strain dependent; therefore, consideration for the complexity of nutrient requirements is essential for improving yields of fungal biocontrol agents.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Effect of nutrient availability on the C, N, and P elemental ratios in the cyanobacterium Microcystis aeruginosa

To clarify whether nutrients limit the growth of Microcystis aeruginosa (Kütz.) Kütz during the growing season in Lake Yogo, we examined the cellular ratios of carbon (C), nitrogen (N), and phosphorus (P) in the populations of M. aeruginosa from August to December 2001. We also measured cellular C, N, and P ratios of M. aeruginosa under batch culture conditions. The cellular levels of N and P of M. aeruginosa in natural population changed more than twofold. The atomic N: C ratio of natural populations of Microcystis fluctuated from 0.11 to 0.26. The atomic P: C ratio fluctuated from 0.0080 to 0.024. The N: C, P: C, and N: P ratios of exponentially growing M. aeruginosa in N-and P-rich medium were 0.19, 0.013, and 15 on average. The growth of M. aeruginosa was suppressed below the N: C ratio of 0.13 under the N-free condition and below the P: C ratio of 0.0026 in the P-free condition. In the natural population, the N: C ratio was low on August 1-2 (0.11) and the P: C ratio was low (less than 0.011) until September. The Microcystis population on August 1-2 was N limited, judging from the results of the culture experiment. In other periods, the population seemed to be supplied with a sufficient amount of N. Although the P: C ratio was low (approximately 0.01) during August and September, it was several times larger than the value of the reduction of growth rate that occurred in culture. P limitation did not occur during the study period. N became more of a limiting factor than P for the formation of blooms of Microcystis. No blooms were observed in August and September, in spite of the increase of cellular levels of N. The formation of Microcystis blooms in Lake Yogo seems to be affected by artificial manipulations such as pumping from Lake Biwa and outflow.     

Influence of growth rate on pigment and lipid composition of the microalgaIsochrysis aff.galbana clone T.iso

A continuous culture ofIsochrysis aff.galbana clone T.iso, used to feedPecten maximus larvae at IFREMER (Brest, France), was carried out in a chemostat at its optimum temperature for growth (26 °C). Changes in pigments, lipid class (neutral, glyco- and phospholipids) and degree of fatty acid unsaturation were studied at three different growth rates (0.33, 0.5, 1 d^–1). As predicted by chemostat theory, a slow growth rate produced higher cell numbers and higher biomass per unit volume. These cells were low in chlorophylla and carotenoids, but rich in neutral lipids. In contrast, cultures with a fast growth rate yielded lower cell concentrations, buth higher chlorophylla, carotenoid and membrane lipid contents per cell. Changes in polyunsaturated fatty acid distribution were related to differences in algal growth rates. Neutral lipids contained mainly saturated and monounsaturated fatty acids (C18:19) at low growth rates whereas they were enriched in polyunsaturated fatty acids, especially C22:63, at high growth rates. Therefore, it is suggested that the growth rate in continuous cultures be controlled so as to adjust the relative proportions of polyunsaturated fatty acids in lipid classes of the diet meant for larval nutrition.Author for correspondence     

VARIATION OF SIPHONAXANTHIN SERIES AMONG THE GENUS NEPHROSELMIS (PRASINOPHYCEAE,CHLOROPHYTA), INCLUDING A NOVEL PRIMARY METHOXY CAROTENOID1

Carotenoid compositions were analyzed for ten strains of Nephroselmis (Prasinophyceae) containing four described and three undescribed species. Based on the distribution pattern of the siphonaxanthin series, five carotenoid types were recognized in the examined strains/species: type I (N. astigmatica Inouye et Pienaar, N. pyriformis (N. Carter) Ettl, and Nephroselmis sp1. MBIC 11158) had siphonaxanthin C12:1 and C14:1 esters as well as 6′‐OH siphonaxanthin C12:1 and C14:1 esters, type II (Nephroselmis sp2. MBIC 11149) had siphonaxanthin C8:1 ester, type III (Nephroselmis sp3. NIES 486, NIES‐PS 535, and MBIC 10871) had 19‐methoxy siphonaxanthin and siphonaxanthin C12:1 and C14:1 esters, type IV (N. spinosa Suda) had only a small amount of siphonaxanthin C12:1 ester, and type V (N. olivacea Stein) had lutein as a major carotenoid but completely lacked the siphonaxanthin series. 19‐Methoxy siphonaxanthin was a novel and very unique carotenoid, that is, it contains a methoxy group and was found for the first time in photosynthetic eukaryotes. Additionally, carotenoids containing a primary methoxy group had previously never been found in any group of organisms. Siphonaxanthin C8:1 ester, which was only known as a trace carotenoid in Chlamydomonas parkeae Ettl, was first discovered as a major carotenoid in Nephroselmis sp2. (MBIC 11149). Based on these results and comparison of the phylogenetic relationships of the Nephroselmis species used, we discuss the taxonomic significance of the carotenoid types and evolutionary process of the photosynthetic antenna systems in green plants.     

Production of single-cell oil from prickly-pear juice fermentation by Cryptococcus curvatus grown in batch culture

The biomass of Cryptococcus curvatus, an oleaginous yeast, reached 11 g/l and accumulated 46% (w/w) lipid when grown for 35 h in batch culture on diluted (25%) prickly-pear juice. The C:N ratio of the juice was about 50 g/g. The efficiency of substrate conversion was 0.48 g/g for biomass and 0.22 g/g for lipids. The extracted lipids were mainly oleic (18:1) and palmitic (16:0) acids and the quality of pipid approached that of palm oil.M. Hassan was and P.J. Blanc, A. Pareilleux and G. Goma are with the Département de Génie Biochimique et Alimentaire, URA-CNRS 544, Institut National des Sciences Appliquées. Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex France; M. Hassan is now with the Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.     

Biological control of strawberry tarsonemid mite Phytonemus pallidus and two-spotted spider mite Tetranychus urticae on strawberry in the UK using species of Neoseiulus (Amblyseius) (Acari: Phytoseiidae)

Two species of Neoseiulus, N. californicus and N. cucumeris, showed potential for biocontrol of phytophagous mites on strawberry. N. californicus controlled Tetranychus urticae on potted strawberry plants in a gauze-sided glasshouse at temperatures comparableto early summer in the UK (8–20°C). Both species of phytoseiid reducednumbers of the tarsonemid Phytonemus pallidus on potted strawberry plants under glasshouse conditions (15–23°C). In several experiments reductions in the range of 71–81% in numbers of tarsonemid active stages and eggs, compared to non-release plants, were obtained. The importance of establishing a suitable predator: prey ratio at an earlystage was demonstrated in an experiment where an initial ratio of 1 N. cucumeris: 10 P. pallidus gave a greater degree of controlthan 1:20 or 1:40.     

Chromoplasts of Tropaeolum majus L.: Lipid synthesis in whole organelles and subfractions

Isolation of tubulous chromoplasts from Tropaeolum majus L. petals was achieved in pure form. Their main substructures-lipid bodies, tubules, and envelope membranes-have been enriched. Whole chromoplasts as well as substructures have been tested for their activities in lipid synthesis. The following activities were found: fatty acid synthesis from acetate, glycosyl transfer reactions from UDP-galactose and UDP-glucose to galactolipids and sterols, acyltransferase reactions from palmitoyl-CoA, and a very active acyl-CoA hydrolase (EC 3.1.2.2.). Fatty acid synthesis was restricted to whole chromoplasts. Glycosyl- and acyltransferases were essentially confined to envelope membranes, whereas acyl-CoA hydrolase was found in all fractions. The chemical composition of chromoplast subfractions was determined. The lipid bodies consisted mainly of galactolipids and carotenoid esters in a 1:1 ratio, together with small amounts of protein.     

Preparation of the red yeast,Xanthophyllomyces dendrorhous,as feed additive with increased availability of astaxanthin

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is used as a colorant for aquaculture, egg yolks, and crustaceans but its carotenoids can only be absorbed by animals when its cell wall is degraded. Conditions that degraded the cell wall of X. dendrorhous were developed. To measure the degrees of cell wall degradation, the carotenoid extractability (extracted carotenoid by acetone/total carotenoid) unit was used. Treatment with HCl (0.2 M, 9 h, 90 °C) followed by neutralization to pH 3 by NaOH and spray-drying increased carotenoid extractability to 100% with minimal decomposition.     

Effects of light and nutrient availability on the growth,allocation, carbon/nitrogen balance,phenolic chemistry,and resistance to herbivory of two freshwater macrophytes

Phenotypic responses of Potamogeton amplifolius and Nuphar advena to different light (7% and 35% of surface irradiance) and nutrient environments were assessed with field manipulation experiments. Higher light and nutrient availability enhanced the growth of P. amplifolius by 154% and 255%, respectively. Additionally, biomass was allocated differently depending on the resource: high light availability resulted in a higher root/shoot ratio, whereas high nutrient availability resulted in a lower root/shoot ratio. Low light availability and high nutrient availability increased the nitrogen content of leaf tissue by 53% and 40% respectively, resulting in a 37% and 31% decrease in the C/N ratio. Root nitrogen content was also increased by low light and high nutrient availability, by 50% (P=0.0807) and 77% respectively, resulting in a 20% and 40% decrease in root C/N ratio. Leaf phenolics were significantly increased 72% by high light and 31% by high nutrient availability, but root phenolic concentrations were not altered significantly. None of these changes in tissue constituents resulted in altered palatability to crayfish. N. advena was killed by the same high nutrient treatment that stimulated growth in P. amplifolius, preventing assessment of phenotypic responses to nutrient availability. However, high light availability increased overall growth by 24%, but this was mainly due to increased growth of the rhizome (increased 100%), resulting in a higher root/shoot ratio. High light tended to increase the production of floating leaves (P=0.09) and significantly decreased the production of submersed leaves. High light availability decreased the nitrogen content by 15% and 25% and increased the phenolic concentration by 88% and 255% in floating and submersed leaves, respectively. These differences in leaf traits did not result in detectable differences in damage by herbivores.     

Physiological responses of Aureoumbra lagunensis and Synechococcus sp. to nitrogen addition in a mesocosm experiment

Aureoumbra lagunensis is the causative organism of the Texas brown tide and is notable because it dominated the Laguna Madre ecosystem from 1990 to 1997. This species is unusual because it has the highest known critical nitrogen to phosphorus ratio (N:P) for any microalgae ranging from 115 to 260, far higher than the 16N:1P Redfield ratio. Because of its high N:P ratio, Aureoumbra should be expected to respond to N additions that would not stimulate the growth of competitors having the Redfield ratio. To evaluate this prediction, a mesocosm experiment was performed in the Laguna Madre, a South Texas coastal lagoon, in which a mixed Aureoumbra–Synechococcus (a cyanobacterium) community was enclosed in 12 mesocosms and subjected to nitrogen addition (6 controls, 6 added ammonium) for 16 days. After day 4, added nitrogen did not significantly increase Aureoumbra specific growth rate but the alga retained dominance throughout the experiment (64–75% of total cell biovolume). In control mesocosms, Aureoumbra became less abundant during the first 4 days of the experiment but rebounded by the end of the experiment and was dominant over Synechococcus. Despite the lack of a strong positive growth response, Aureoumbra did respond physiologically to N addition. By the end of the experiment, the average N:P ratio of the Aureoumbra-dominated community was 86 in the N+ treatment and 41 in the control, indicating that the alga became less N-limited in the N+ treatment. The average C:N ratio was 6.6 in the N+ treatment (8.6 in the control) and suggests that the alga was not N-limited, however, C:N ratio may not be a good indicator of nitrogen limitation since this alga can produce significant quantities of carbon-containing extracellular polysaccharides, depending on growth conditions. Both Aureoumbra cellular chlorophyll fluorescence and cell size increased in response to added N, indicating a reduction in N limitation. It appeared that the N additions were not large and/or frequent enough to stimulate Aureoumbra growth. The main competitor, the unicellular cyanobacterium Synechococcus, responded positively to the nitrogen addition by increased specific growth rate. Unlike Aureoumbra, no significant effect on Synechococcus cellular pigment fluorescence or cell size was noted. Literature data suggest that Synechococcus, like Aureoumbra, may have a critical N:P ratio much higher than 16:1, which could explain its response.