The tiny giant of the sea, Ostreococcus's unique adaptations.

Ostreococcus spp. are unicellular organisms with one of the simplest cellular organizations. The sequencing of the genomes of different Ostreococcus species has reinforced this status since Ostreococcus tauri has one most compact nuclear genomes among eukaryotic organisms. Despite this, it has retained a number of genes, setting it apart from other organisms with similar small genomes. Ostreococcus spp. feature a substantial number of selenocysteine-containing proteins, which, due to their higher catalytic activity compared to their selenium-lacking counterparts, may require a reduced quantity of proteins. Notably, O. tauri encodes several ammonium transporter genes, that may provide it with a competitive edge for acquiring nitrogen (N). This characteristic makes it an intriguing model for studying the efficient use of N in eukaryotes. Under conditions of low N availability, O. tauri utilizes N from abundant proteins or amino acids, such as L-arginine, similar to higher plants. However, the presence of a nitric oxide synthase (L-arg substrate) sheds light on a new metabolic pathway for L-arg in algae. The metabolic adaptations of O. tauri to day and night cycles offer valuable insights into carbon and iron metabolic configuration. O. tauri has evolved novel strategies to optimize iron uptake, lacking the classic components of the iron absorption mechanism. Overall, the cellular and genetic characteristics of Ostreococcus contribute to its evolutionary success, making it an excellent model for studying the physiological and genetic aspects of how green algae have adapted to the marine environment. Furthermore, given its potential for lipid accumulation and its marine habitat, it may represent a promising avenue for third-generation biofuels.

Three step flow focusing enables image-based discrimination and sorting of late stage 1 Haematococcus pluvialis cells.

Label-free and gentle separation of cell stages with desired target properties from mixed stage populations are a major research task in modern biotechnological cultivation process and optimization of micro algae. The reported microfluidic sorter system (MSS) allows the subsequent investigation of separated subpopulations. The implementation of a viability preserving MSS is shown for separation of late stage 1 Haematococcus pluvialis (HP) cells form a mixed stage population. The MSS combines a three-step flow focusing unit for aligning the cells in single file transportation mode at the center of the microfluidic channel with a pure hydrodynamic sorter structure for cell sorting. Lateral displacement of the cells into one of the two outlet channels is generated by piezo-actuated pump chambers. In-line decision making for sorting is based on a user-definable set of image features and properties. The reported MSS significantly increased the purity of target cells in the sorted population (94%) in comparison to the initial mixed stage population (19%).

Double-high in palmitic and oleic acids accumulation in a non-model green microalga, Messastrum gracile SE-MC4 under nitrate-repletion and -starvation cultivations.

Bioprospecting for biodiesel potential in microalgae primarily involves a few model species of microalgae and rarely on non-model microalgae species. Therefore, the present study determined changes in physiology, oil accumulation, fatty acid composition and biodiesel properties of a non-model microalga Messastrum gracile SE-MC4 in response to 12 continuous days of nitrate-starve (NS) and nitrate-replete (NR) conditions respectively. Under NS, the highest oil content (57.9%) was achieved despite reductions in chlorophyll content, biomass productivity and lipid productivity. However, under both NS and NR, palmitic acid and oleic acid remained as dominant fatty acids thus suggesting high potential of M. gracile for biodiesel feedstock consideration. Biodiesel properties analysis returned high values of cetane number (CN 61.9-64.4) and degree of unsaturation (DU 45.3-57.4) in both treatments. The current findings show the possibility of a non-model microalga to inherit superior ability over model species in oil accumulation for biodiesel development.

Response of Pseudokirchneriella subcapitata in Free and Alginate Immobilized Cells to Heavy Metals Toxicity.

Effects of 12 heavy metals on growth of free and alginate-immobilized cells of the alga Pseudokirchneriella subcapitata were investigated. The tested metals ions include Al, As, Cd, Co, Cr, Cu, Hg, Se, Ni, Pb, Sr, and Zn. Toxicity values (EC50) were calculated by graphical interpolation from dose-response curves. The highest to the lowest toxic metals are in the order Cd > Co > Hg > Cu > Ni > Zn > Cr > Al > Se > As > Pb > Sr. The lowest metal concentration (mg L-1) inhibiting 50% (EC50) of algal growth of free and immobilized (values in parentheses) algal cells were, 0.018 (0.09) for Cd, 0.03 (0.06) for Co, 0.039 (0.06) for Hg, 0.048 (0.050) for Cu, 0.055 (0.3) for Ni, 0.08 (0.1) for Zn, 0.2 (0.3) for Cr, 0.75 (1.8) for Al, 1.2 (1.4) for Se, 3.0 (4.0) for As, 3.3 (5.0) for Pb, and 160 (180) for Sr. Free and immobilized cultures showed similar responses to Cu and Se. The free cells were more sensitive than the immobilized ones. Accordingly, the toxicity (EC50) of heavy metals derived only form immobilized algal cells might by questionable. The study suggests that batteries of alginate-immobilized algae can efficiently replace free algae for the bio-removal of heavy metals.

From algal cells to autofluorescent ghost plasma membrane vesicles.

Plasma membrane vesicles can be effective, non-toxic carriers for microscale material transport, provide a convenient model for probing membrane-related processes, since intracellular biochemical processes are eliminated. We describe here a fine-tuned protocol for isolating ghost plasma membrane vesicles from the unicellular alga Dunaliella tertiolecta, and preliminary characterization of their structural features and permeability properties, with comparisons to giant unilamellar phospholipid vesicles. The complexity of the algal ghost membrane vesicles reconstructed from the native membrane material released after hypoosmotic stress lies between that of phospholipid vesicles and cells. AFM structural characterization of reconstructed vesicles shows a thick envelope and a nearly empty vesicle interior. The surface of the envelope contains a heterogeneous distribution of densely packed, nanometer-scale globules and pore-like structures which may be derived from surface coat proteins. Confocal fluorescence imaging reveals the highly pigmented photosynthetic apparatus located within the thylakoid membrane and retained in the vesicle membrane. Transport of the fluorescent dye calcein into ghost and giant unilamellar vesicles reveals significant differences in permeability. Expanded knowledge of this unique membrane system will contribute to the design of marine bio-inspired carriers for advanced biotechnological applications.

Mechanical stress induced astaxanthin accumulation of H. pluvialis on a chip.

Microalgae have been envisioned as a source of food, feed, health nutraceuticals, and cosmetics. Among various microalgae, Haematococcus pluvialis (H. pluvialis) is known to be the richest feedstock of natural astaxanthin. Astaxanthin is a highly effective antioxidation material and is being widely used in aquaculture, nutraceuticals, pharmacology, and feed industries. Here, we present a microfluidic chip consisting of a micropillar array and six sets of culture chambers, which enables sorting of motile flagellated vegetative stage H. pluvialis (15-20 µm) from cyst stage H. pluvialis as well as culture of the selected cells under a mechanically stressed microenvironment. The micropillar array successfully sorted only the motile early vegetative stage cells (avg. size = 19.8 ± 1.6 µm), where these sorted cells were uniformly loaded inside each culture chamber (229 ± 39 cells per chamber). The mechanical stress level applied to the cells was controlled by designing the culture chambers with different heights (5-70 µm). Raman analysis results revealed that the mechanical stress indeed induced the accumulation of astaxanthin in H. pluvialis. Also, the most effective chamber height enhancing the astaxanthin accumulation (i.e., 15 µm) was successfully screened using the developed chip. Approximately 9 times more astaxanthin accumulation was detected after 7 days of culture compared to the no mechanical stress condition. The results clearly demonstrate the capability of the developed chip to investigate bioactive metabolite accumulation of microalgae induced by mechanical stress, where the amount was quantitatively analyzed in a label-free manner. We believe that the developed chip has great potential for studying the effects of mechanical stress on not only H. pluvialis but also various microalgal species in general.

Pediludiella daitoensis gen. et sp. nov. (Scenedesmaceae, Chlorophyceae), a large coccoid green alga isolated from a Loxodes ciliate.

Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039 and other non-floating green microalgae revealed some of the unique gene families belonging to this floating microalga, which may be involved in biofilm formation. Furthermore, genetic transformation of this microalga was achieved with an electroporation method. The genome information and transformation techniques presented in this study will be useful to obtain molecular insights into the water surface-floating phenotype of Chlorococcum sp. FFG039.

Chronic exposure of the freshwater alga Pseudokirchneriella subcapitata to five oxide nanoparticles: Hazard assessment and cytotoxicity mechanisms.

The increasing use of nanoparticles (NPs) unavoidably enhances their unintended introduction into the aquatic systems, raising concerns about their nanosafety. This work aims to assess the toxicity of five oxide NPs (Al2O3, Mn3O4, In2O3, SiO2 and SnO2) using the freshwater alga Pseudokirchneriella subcapitata as a primary producer of ecological relevance. These NPs, in OECD medium, were poorly soluble and unstable (displayed low zeta potential values and presented the tendency to agglomerate). Using the algal growth inhibition assay and taking into account the respective 72 h-EC50 values, it was possible to categorize the NPs as: toxic (Al2O3 and SnO2); harmful (Mn3O4 and SiO2) and non-toxic/non-classified (In2O3). The toxic effects were mainly due to the NPs, except for SnO2 which toxicity can mainly be attributed to the Sn ions leached from the NPs. A mechanistic study was undertaken using different physiological endpoints (cell membrane integrity, metabolic activity, photosynthetic efficiency and intracellular ROS accumulation). It was observed that Al2O3, Mn3O4 and SiO2 induced an algistatic effect (growth inhibition without loss of membrane integrity) most likely as a consequence of the cumulative effect of adverse outcomes: i) reduction of the photosynthetic efficiency of the photosystem II (ФPSII); ii) intracellular ROS accumulation and iii) loss of metabolic activity. SnO2 NPs also provoked an algistatic effect probably as a consequence of the reduction of ФPSII since no modification of intracellular ROS levels and metabolic activity were observed. Altogether, the results here presented allowed to categorize the toxicity of the five NPs and shed light on the mechanisms behind NPs toxicity in the green alga P. subcapitata.

The selection of a surfactant for freshwater microalgae harvesting and separation by the foam separation method.

Collecting microalgae from water with less energy and cost is significant to gain economic profit from microalgae harvesting and processing. Foam separation has certain advantages including low energy consumption, simple operation and easy maintenance of the equipment. Natural surfactants, compared to traditional surfactants, were used to harvest and separate the freshwater microalgae Desmodesmus brasiliensis by foam separation. Results showed a recovery percentage of 93.6% and an enrichment ratio of 23.1 with the natural surfactant cocamidopropyl betaine (CAPB), suggesting that this low-cost surfactant can be applied to microalgae biomass recovery on a commercial scale using foam separation with no negative effect on the content of microalgae chlorophyll, carotenoid or protein.

Size-abundance rules? Evolution changes scaling relationships between size, metabolism and demography.

Body size often strongly covaries with demography across species. Metabolism has long been invoked as the driver of these patterns, but tests of causal links between size, metabolism and demography within a species are exceedingly rare. We used 400 generations of artificial selection to evolve a 2427% size difference in the microalga Dunaliella tertiolecta. We repeatedly measured size, energy fluxes and demography across the evolved lineages. Then, we used standard metabolic theory to generate predictions of how size and demography should covary based on the scaling of energy fluxes that we measured. The size dependency of energy remained relatively consistent in time, but metabolic theory failed to predict demographic rates, which varied unpredictably in strength and even sign across generations. Classic theory holds that size affects demography via metabolism - our results suggest that both metabolism and size act separately to drive demography and that among-species patterns may not predict within-species processes.

Changes in nanomechanical properties and adhesion dynamics of algal cells during their growth.

Nanomechanical and structural characterisations of algal cells are of key importance for understanding their adhesion behaviour at interfaces in the aquatic environment. We examine here the nanomechanical properties and adhesion dynamics of the algal cells during two phases of their growth using complementary surface methods and the mathematical modelling. Mechanical properties of motile cells are hard to assess while keeping cells viable, and studies to date have been limited. Immobilisation of negatively charged cells to a positively charged substrate enables high-resolution AFM imaging and nanomechanical measurements. Cells were stiffer and more hydrophobic in the exponential than in the stationary phase, suggesting molecular modification of the cell envelope during aging. The corresponding properties of algal cells were in agreement with the increase of critical interfacial tensions of adhesion, determined amperometrically. Cells in exponential phase possessed a larger cell volume, in agreement with the large amount of amperometrically measured displaced charge at the interface. Differences in the kinetics of adhesion and spreading of cells at the interface were attributed to their various volumes and nanomechanical properties that varied during cell aging. Our findings contribute to the present body of knowledge on the biophysics of algal cells on a fundamental level.

Algal cell response to laboratory-induced cadmium stress: a multimethod approach.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Evaluation and optimization of a methodology for the long-term cryogenic storage of Tetradesmus obliquus at - 80 °C.

Cryopreservation is a common methodology for long-term microalgae storage. Current cryopreservation methods are based on using diverse cryoprotectants and two-step cooling protocols, followed by sample storage at the temperature of liquid nitrogen (- 196 °C). However, the use of this methodology requires a continuous liquid N2 supply as well as facilities with dedicated equipment, which is not affordable for every laboratory. In our work, we report on the successful development of a simple and cost-effective method for the long-term cryogenic storage of Tetradesmus obliquus at temperatures (- 80 °C) used in commonly available deep freezers that are more readily accessible to laboratories. Two procedures were evaluated that were originally devised for other microalgae; this was followed by the optimization of critical parameters such as the sample's microalgal concentration and the cryoprotectant reagent's incubation time. Cell viability was monitored using the survival rates obtained by direct agar plating and the growth recovery times in liquid cultures. Viability-related variables were recorded following different storage times of up to 3 years. The main operational factors involved in the process (cell concentration, incubation time, and storage time) were statistically analyzed with regard to their influence on the survival rate. The statistical analysis showed interdependence (a two-factor interaction) between the cellular concentration and the cryoprotectant's incubation time, on the one hand, and between the incubation time and the storage time on the other. Survival rates above 70% were obtained under optimized conditions after 3 months of storage, along with 20-35% viabilities after 3 years. These results open up the possibility of extending this method to other Scenedesmaceae, or even other microalgal species, and for its use in resource-limited laboratories.

Two-Stage Cultivation of Dunaliella tertiolecta with Glycerol and Triethylamine for Lipid Accumulation: a Viable Way To Alleviate the Inhibitory Effect of Triethylamine on Biomass.

Microalgae are promising alternatives for sustainable biodiesel production. Previously, it was found that 100 ppm triethylamine greatly enhanced lipid production and lipid content per cell of Dunaliella tertiolecta by 20% and 80%, respectively. However, triethylamine notably reduced biomass production and pigment contents. In this study, a two-stage cultivation with glycerol and triethylamine was attempted to improve cell biomass and lipid accumulation. At the first stage with 1.0 g/liter glycerol addition, D. tertiolecta cells reached the late log phase in a shorter time due to rapid cell growth, leading to the highest cell biomass (1.296 g/liter) for 16 days. However, the increased glycerol concentrations with glycerol addition decreased the lipid content. At the second-stage cultivation with 100 ppm triethylamine, the highest lipid concentration and lipid weight content were 383.60 mg/liter and 37.7% of dry cell weight (DCW), respectively, in the presence of 1.0 g/liter glycerol, which were 27.36% and 72.51% higher than those of the control group, respectively. Besides, the addition of glycerol alleviated the inhibitory effect of triethylamine on cell morphology, algal growth, and pigment accumulation in D. tertiolecta The results indicated that two-stage cultivation is a viable way to improve lipid yield in microalgae.IMPORTANCE Microalgae are promising alternatives for sustainable biodiesel production. Two-stage cultivation with glycerol and triethylamine enhanced the lipid productivity of Dunaliella tertiolecta, indicating that two-stage cultivation is an efficient strategy for biodiesel production from microalgae. It was found that glycerol significantly enhanced cell biomass of D. tertiolecta, and the presence of glycerol alleviated the inhibitory effect of triethylamine on algal growth. Glycerol, the major byproduct from biodiesel production, was used for the biomass accumulation of D. tertiolecta at the first stage of cultivation. Triethylamine, as a lipid inducer, was used for lipid accumulation at the second stage of cultivation. Two-stage cultivation with glycerol and triethylamine enhanced lipid productivity and alleviated the inhibitory effect of triethylamine on the algal growth of D. tertiolecta, which is an efficient strategy for lipid production from D. tertiolecta.

Enhancing Astaxanthin Accumulation in Haematococcus pluvialis by Coupled Light Intensity and Nitrogen Starvation in Column Photobioreactors.

Natural astaxanthin mainly derives from a microalgae producer, Haematococcus pluvialis. The induction of nitrogen starvation and high light intensity is particularly significant for boosting astaxanthin production. However, the different responses to light intensity and nitrogen starvation needed to be analyzed for biomass growth and astaxanthin accumulation. The results showed that the highest level of astaxanthin production was achieved in nitrogen starvation, and was 1.64 times higher than the control group at 11 days. With regard to the optimization of light intensity utilization, it was at 200 µmo/m²/s under nitrogen starvation that the highest astaxanthin productivity per light intensity was achieved. In addition, both high light intensity and a nitrogen source had significant effects on multiple indicators. For example, high light intensity had a greater significant effect than a nitrogen source on biomass dry weight, astaxanthin yield and astaxanthin productivity; in contrast, nitrogen starvation was more beneficial for enhancing astaxanthin content per dry weight biomass. The data indicate that high light intensity synergizes with nitrogen starvation to stimulate the biosynthesis of astaxanthin.

Do larger individuals cope with resource fluctuations better? An artificial selection approach.

Size determines the rate at which organisms acquire and use resources but it is unclear what size should be favoured under unpredictable resource regimes. Some theories claim smaller organisms can grow faster following a resource pulse, whereas others argue larger species can accumulate more resources and maintain growth for longer periods between resource pulses. Testing these theories has relied on interspecific comparisons, which tend to confound body size with other life-history traits. As a more direct approach, we used 280 generations of artificial selection to evolve a 10-fold difference in mean body size between small- and large-selected phytoplankton lineages of Dunaliella tertiolecta, while controlling for biotic and abiotic variables. We then quantified how body size affected the ability of this species to grow at nutrient-replete conditions and following periods of nitrogen or phosphorous deprivation. Overall, smaller cells showed slower growth, lower storage capacity and poorer recovery from phosphorous depletion, as predicted by the 'fasting endurance hypothesis'. However, recovery from nitrogen limitation was independent of size-a finding unanticipated by current theories. Phytoplankton species are responsible for much of the global carbon fixation and projected trends of cell size decline could reduce primary productivity by lowering the ability of a cell to store resources.

Quantification of cyanobacterial cells via a novel imaging-driven technique with an integrated fluorescence signature.

A novel imaging-driven technique with an integrated fluorescence signature to enable automated enumeration of two species of cyanobacteria and an alga of somewhat similar morphology to one of the cyanobacteria is presented to demonstrate proof-of-concept that high accuracy, imaging-based, rapid water quality analysis can be with conventional equipment available in typical water quality laboratories-this is not currently available. The results presented herein demonstrate that the developed method identifies and enumerates cyanobacterial cells at a level equivalent to or better than that achieved using standard manual microscopic enumeration techniques, but in less time, and requiring significantly fewer resources. When compared with indirect measurement methods, the proposed method provides better accuracy at both low and high cell concentrations. It extends the detection range for cell enumeration while maintaining accuracy and increasing enumeration speed. The developed method not only accurately estimates cell concentrations, but it also reliably distinguishes between cells of Anabaena flos-aquae, Microcystis aeruginosa, and Ankistrodesmus in mixed cultures by taking advantage of additional contrast between the target cell and complex background gained under fluorescent light. Thus, the proposed image-driven approach offers promise as a robust and cost-effective tool for identifying and enumerating microscopic cells based on their unique morphological features.

Chloromonas svalbardensis n. sp. with Insights into the Phylogroup Chloromonadinia (Chlorophyceae).

The traditional green algal genus Chloromonas accommodates mesophilic, cold-tolerant and cold-adapted microorganisms. In this paper, we studied a new strain isolated from a wet hummock meadow in the High Arctic. We used morphological, ultrastructural and molecular data to assess the taxonomic position and phylogenetic relationships of the new isolate. The observed morphological features generally corresponded to the cold-tolerant Chloromonas characteristics. However, ellipsoidal or wide ellipsoidal vegetative cells, a massive parietal cup-shaped chloroplast with a number of continuously connected lobes, a thick cell wall, a prominent hemispherical papilla and the anterior position of an oblong or round eyespot distinguished the alga from all previously described Chloromonas species. Analyses of rbcL and 18S rRNA genes showed that the new strain formed an independent lineage within a clade containing mesophilic and psychrotolerant Chloromonas species. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker supported a separate species identity of the new isolate. Considering the morphological and molecular differences from its relatives, a new psychrotolerant species, Chloromonas svalbardensis, is proposed. Further, our results demonstrated the paraphyletic origin of Chloromonas within Chloromonadinia with genetically, morphologically and ecologically well-defined clades. We discuss a scenario of a possible Chloromonas split and revision.

Cell size, photosynthesis and the package effect: an artificial selection approach.

Cell size correlates with most traits among phytoplankton species. Theory predicts that larger cells should show poorer photosynthetic performance, perhaps due to reduced intracellular self-shading (i.e. package effect). Yet current theory relies heavily on interspecific correlational approaches and causal relationships between size and photosynthetic machinery have remained untested. As a more direct test, we applied 250 generations of artificial selection (c. 20 months) to evolve the green microalga Dunaliella teriolecta (Chlorophyta) toward different mean cell sizes, while monitoring all major photosynthetic parameters. Evolving larger sizes (> 1500% difference in volume) resulted in reduced oxygen production per chlorophyll molecule - as predicted by the package effect. However, large-evolved cells showed substantially higher rates of oxygen production - a finding unanticipated by current theory. In addition, volume-specific photosynthetic pigments increased with size (Chla+b), while photo-protectant pigments decreased (ß-carotene). Finally, larger cells displayed higher growth performances and Fv /Fm , steeper slopes of rapid light curves (α) and smaller light-harvesting antennae (σPSII ) with higher connectivity (ρ). Overall, evolving a common ancestor into different sizes showed that the photosynthetic characteristics of a species coevolves with cell volume. Moreover, our experiment revealed a trade-off between chlorophyll-specific (decreasing with size) and volume-specific (increasing with size) oxygen production in a cell.