Thylakoid ultrastructural variations in chlorophyll-deficient wheat: aberrations or structural acclimation?

MAIN CONCLUSION: A structural re-modeling of the thylakoid system, including granum size and regularity, occurs in chlorophyll-deficient wheat mutants affected by photosynthetic membrane over-reduction. In the chloroplast of land plants, the thylakoid system is defined by appressed grana stacks and unstacked stroma lamellae. This study focuses on the variations of the grana organization occurring in outdoor-grown wheat mutants characterized by low chlorophyll content and a tendency for photosynthetic membrane over-reduction. Triticum aestivum ANK-32A and Triticum durum ANDW-7B were compared to their corresponding WT lines, NS67 and LD222, respectively. Electron micrographs of chloroplasts were used to calculate grana ultrastructural parameters. Photosynthetic parameters were obtained by modulated chlorophyll fluorescence and applying Light Curves (LC) and Rapid Light Curves (RLC) protocols. For each photosynthetic parameter, the difference Δ(RLC-LC) was calculated to evaluate the flexible response to light in the examined lines. In the mutants, fewer and smaller disks formed grana stacks characterized by a marked increase in lateral and cross-sectional irregularity, both negatively correlated with the number of layers per granum. A relationship was found between membrane over-reduction and granum structural irregularity. The possible acclimative significance of a greater proportion of stroma-exposed grana domains in relieving the excess electron pressure on PSI is discussed.

Ultrastructural organization of the thylakoid system during the afternoon relocation of the giant chloroplast in Selaginella martensii Spring (Lycopodiophyta).

Within the ancient vascular plant lineage known as lycophytes, many Selaginella species contain only one giant chloroplast in the upper epidermal cells of the leaf. In deep-shade species, such as S. martensii, the chloroplast is cup-shaped and the thylakoid system differentiates into an upper lamellar region and a lower granal region (bizonoplast). In this report, we describe the ultrastructural changes occurring in the giant chloroplast hosted in the epidermal cells of S. martensii during the daily relocation of the organelle. The process occurs in up to ca. 40% of the microphylls without the plants being exposed to high-light flecks. The relocated chloroplast loses its cup shape: first, it flattens laterally toward the radial cell wall and then assumes a more globular shape. The loss of the conical cell shape, the side-by-side lateral positioning of vacuole and chloroplast, and the extensive rearrangement of the thylakoid system to only granal cooperate in limiting light absorption. While the cup-shaped chloroplast emphasizes the light-harvesting capacity in the morning, the relocated chloroplast is suggested to support the renewal of the thylakoid system during the afternoon, including the recovery of photosystem II (PSII) from photoinhibition. The giant chloroplast repositioning is part of a complex reversible reshaping of the whole epidermal cell.

Thylakoid membrane appression in the giant chloroplast of Selaginella martensii Spring: A lycophyte challenges grana paradigms in shade-adapted species.

In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.

Enhancing Urban Wastewater Treatment through Isolated Chlorella Strain-Based Phytoremediation in Centrate Stream: An Analysis of Algae Morpho-Physiology and Nutrients Removal Efficiency.

The release of inadequately treated urban wastewater is the main cause of environmental pollution of aquatic ecosystems. Among efficient and environmentally friendly technologies to improve the remediation process, those based on microalgae represent an attractive alternative due to the potential of microalgae to remove nitrogen (N) and phosphorus (P) from wastewaters. In this work, microalgae were isolated from the centrate stream of an urban wastewater treatment plant and a native Chlorella-like species was selected for studies on nutrient removal from centrate streams. Comparative experiments were set up using 100% centrate and BG11 synthetic medium, modified with the same N and P as the effluent. Since microalgal growth in 100% effluent was inhibited, cultivation of microalgae was performed by mixing tap-freshwater with centrate at increasing percentages (50%, 60%, 70%, and 80%). While algal biomass and nutrient removal was little affected by the differently diluted effluent, morpho-physiological parameters (FV/FM ratio, carotenoids, chloroplast ultrastructure) showed that cell stress increased with increasing amounts of centrate. However, the production of an algal biomass enriched in carotenoids and P, together with N and P abatement in the effluent, supports promising microalgae applications that combine centrate remediation with the production of compounds of biotechnological interest; for example, for organic agriculture.

Long-Term Alleviation of the Functional Phenotype in Chlorophyll-Deficient Wheat and Impact on Productivity: A Semi-Field Phenotyping Experiment.

Wheat mutants with a reduced chlorophyll synthesis are affected by a defective control of the photosynthetic electron flow, but tend to recover a wild-type phenotype. The sensitivity of some mutants to light fluctuations suggested that cultivation outdoors could significantly impact productivity. Six mutant lines of Triticum durum or Triticum aestivum with their respective wild-type cultivars were cultivated with a regular seasonal cycle (October-May) in a semi-field experiment. Leaf chlorophyll content and fluorescence parameters were analysed at the early (November) and late (May) developmental stages, and checked for correlation with morphometric and grain-production parameters. The alleviation of the phenotype severity concerned primarily the recovery of the photosynthetic-membrane functionality, but not the leaf chlorophyll content. Photosystem II (PSII) was less photoprotected in the mutants, but a moderate PSII photoinhibition could help control the electron flow into the chain. The accumulation of interchain electron carriers was a primary acclimative response towards the naturally fluctuating environment, maximally exploited by the mature durum-wheat mutants. The mutation itself and/or the energy-consuming compensatory mechanisms markedly influenced the plant morphogenesis, leading especially to reduced tillering, which in turn resulted in lower grain production per plant. Consistently with the interrelation between early photosynthetic phenotype and grain-yield per plant, chlorophyll-fluorescence indexes related to the level of photoprotective thermal dissipation (pNPQ), photosystem II antenna size (ABS/RC), and pool of electron carriers (Sm) are proposed as good candidates for the in-field phenotyping of chlorophyll-deficient wheat.

Fast chlorophyll a fluorescence induction (OJIP) phenotyping of chlorophyll-deficient wheat suggests that an enlarged acceptor pool size of Photosystem I helps compensate for a deregulated photosynthetic electron flow.

The wheat lines affected by a decrease in the leaf chlorophyll content typically experience a biomass loss. A known major problem of the chlorophyll-deficient wheat mutants is their limited prevention of Photosystem I (PSI) over-reduction brought about by an insufficient cyclic electron flow, potentially exposing them to a higher sensitivity to light fluctuations. However, the resistance of some mutant lines against fluctuating light suggests the occurrence of regulatory processes compensating for the defect in cyclic electron flow. In this study, a phenotyping approach based on fast chlorophyll a fluorescence induction (OJIP transient), corroborated by P700 redox kinetics, was applied to a collection of chlorophyll-deficient wheat lines, grown under continuous or fluctuating light. Quantitative parameters calculated from the OJIP transient are considered informative about Photosystem II (PSII) functional antenna size and photochemistry, as well as the functioning of the entire photosynthetic electron transport chain. The mutants tended to recover a wild-type-like chlorophyll content, and mature plants could hardly be distinguished based on their effective PSII antenna size. Nevertheless, specific OJIP-derived parameters were strongly correlated with the phenotype severity, in particular the amplitude of the I-P phase and the I-P/J-P amplitude ratio, which are indicative of a more capacitive pool of PSI final electron acceptors (ferredoxin and ferredoxin-NADP+ oxidoreductase, FNR). We propose that the enlargement of such pool of electron carriers is a compensatory response operating at the acceptor side of PSI to alleviate potentially harmful over-reduced states of PSI. Our results also suggest that, in chlorophyll-deficient mutants, higher FV /FM cannot prove a superior PSII photochemistry and wider I-P phase is not indicative of a higher relative content of PSI.

Photosystem II photoinhibition and photoprotection in a lycophyte, Selaginella martensii.

The Lycophyte Selaginella martensii efficiently acclimates to diverse light environments, from deep shade to full sunlight. The plant does not modulate the abundance of the Light Harvesting Complex II, mostly found as a free trimer, and does not alter the maximum capacity of thermal dissipation (NPQ). Nevertheless, the photoprotection is expected to be modulatable upon long-term light acclimation to preserve the photosystems (PSII, PSI). The effects of long-term light acclimation on PSII photoprotection were investigated using the chlorophyll fluorometric method known as "photochemical quenching measured in the dark" (qPd ). Singularly high-qPd values at relatively low irradiance suggest a heterogeneous antenna system (PSII antenna uncoupling). The extent of antenna uncoupling largely depends on the light regime, reaching the highest value in sun-acclimated plants. In parallel, the photoprotective NPQ (pNPQ) increased from deep-shade to high-light grown plants. It is proposed that the differences in the long-term modulation in the photoprotective capacity are proportional to the amount of uncoupled LHCII. In deep-shade plants, the inconsistency between invariable maximum NPQ and lower pNPQ is attributed to the thermal dissipation occurring in the PSII core.

Characterization of Neochloris oleoabundans under Different Cultivation Modes and First Results on Bioactivity of Its Extracts against HCoV-229E Virus.

Microalgae are proposed in several biotechnological fields because of their ability to produce biomass enriched in high-value compounds according to cultivation conditions. Regarding the health sector, an emerging area focuses on natural products exploitable against viruses. This work deals with the characterization of the green microalga Neochloris oleoabundans cultivated under autotrophic and mixotrophic conditions as a source of whole aqueous extracts, tested as antivirals against HCoV-229E (Coronaviridae family). Glucose was employed for mixotrophic cultures. Growth and maximum quantum yield of photosystem II were monitored for both cultivations. Algae extracts for antiviral tests were prepared using cultures harvested at the early stationary phase of growth. Biochemical and morphological analyses of algae indicated a different content of the most important classes of bioactive compounds with antiviral properties (lipids, exo-polysaccharides, and total phenolics, proteins and pigments). To clarify which phase of HCoV-229E infection on MRC-5 fibroblast cells was affected by N. oleoabundans extracts, four conditions were tested. Extracts gave excellent results, mainly against the first steps of virus infection. Notwithstanding the biochemical profile of algae/extracts deserves further investigation, the antiviral effect may have been mainly promoted by the combination of proteins/pigments/phenolics for the extract derived from autotrophic cultures and of proteins/acidic exo-polysaccharides/lipids in the case of mixotrophic ones.

Increased photosynthesis from a deep-shade to high-light regime occurs by enhanced CO2 diffusion into the leaf of Selaginella martensii.

The current understanding of photosynthesis across land plant phylogeny strongly indicates that ancient vascular plants are mainly limited by strong constitutive CO2 diffusional constraints, particularly low stomatal and mesophyll conductance. Considering that the lycophyte Selaginella martensii can demonstrate long-term light acclimation, this study addresses the regulation extent of CO2 assimilation in this species cultivated under contrasting light regimes of deep shade, medium shade and high light. Comparative analyses of photosynthetic traits, CO2 conductance and leaf morpho-anatomy revealed acclimation plasticity similar to that of seed plants, though occurring in the context of an inherently low photosynthetic capacity typical of lycophytes. Specific modulations of the stomatal density and aperture, chloroplast surface exposed to mesophyll airspaces and cell wall thickness sustained a marked improvement in CO2 diffusion from deep shade to high light. However, the maximum carboxylation rate was comparatively less effectively upregulated, leading to a greater incidence of biochemical limitations of photosynthesis. Because of a low carboxylation capacity under any light regime, a lycophyte prevents potential photodamage to the chloroplast by not only exploiting the thermal dissipation of excess absorbed energy but also diverting a large fraction of photosynthetic electrons to sinks alternative to carboxylation.

In an ancient vascular plant the intermediate relaxing component of NPQ depends on a reduced stroma: Evidence from dithiothreitol treatment.

In plants, the non-photochemical quenching of chlorophyll fluorescence (NPQ) induced by high light reveals the occurrence of a multiplicity of regulatory processes of photosynthesis, primarily devoted to photoprotection of photosystem I and II (PSI and PSII). The study of NPQ relaxation in darkness allows the separation of three kinetically distinct phases: the fast relaxing high-energy quenching qE, the intermediate relaxing phase and the nearly non-relaxatable photoinhibitory quenching. Several processes can underlie the intermediate phase. In the ancient vascular plant Selaginella martensii (Lycopodiophyta) this component, here termed qX, was previously proposed to reflect mainly a photoprotective energy-spillover from PSII to PSI. It is hypothesized that qX is induced by an over-reduced photosynthetic electron transport chain from PSII to final acceptors. To test this hypothesis the leaves were treated with the reductant dithiothreitol (DTT) and the chlorophyll fluorescence changes were analysed during the induction with high irradiance and the subsequent relaxation in darkness. DTT treatment caused the well-known decrease in NPQ induction and expectedly resulted in a disturbed photosynthetic electron flow. The relaxation curves of Y(NPQ), formally representing the quantum yield of the regulatory thermal dissipation, revealed a DTT dose-dependent decrease in amplitude not only of qE, but also of qX, up to the complete disappearance of the latter. Modelling of the relaxation curves under alternative scenarios led to the conclusion that DTT is permissive with respect to qX induction but suppresses its dark relaxation. The strong dependence of qX on the chloroplast redox state is discussed with respect to its proposed energy-spillover photoprotective significance in a lycophyte.

Removal of Nitrogen and Phosphorus from Thickening Effluent of an Urban Wastewater Treatment Plant by an Isolated Green Microalga.

Microalgae are photosynthetic microorganisms and are considered excellent candidates for a wide range of biotechnological applications, including the removal of nutrients from urban wastewaters, which they can recover and convert into biomass. Microalgae-based systems can be integrated into conventional urban wastewater treatment plants (WW-TP) to improve the water depuration process. However, microalgal strain selection represents a crucial step for effective phytoremediation. In this work, a microalga isolated from the effluent derived from the thickening stage of waste sludge of an urban WW-TP was selected and tested to highlight its potential for nutrient removal. Ammonium and phosphate abatements by microalgae were evaluated using both the effluent and a synthetic medium in a comparative approach. Parallelly, the isolate was characterized in terms of growth capability, morphology, photosynthetic pigment content and photosystem II maximum quantum yield. The isolated microalga showed surprisingly high biomass yield and removal efficiency of both ammonium and phosphate ions from the effluent but not from the synthetic medium. This suggests its clear preference to grow in the effluent, linked to the overall characteristics of this matrix. Moreover, biomass from microalgae cultivated in wastewater was enriched in photosynthetic pigments, polyphosphates, proteins and starch, but not lipids, suggesting its possible use as a biofertilizer.

Biological aspects and biotechnological potential of marine diatoms in relation to different light regimens.

As major primary producers in marine environments, diatoms are considered a valuable feedstock of biologically active compounds for application in several biotechnological fields. Due to their metabolic plasticity, especially for light perception and use and in order to make microalgal production more environmentally sustainable, marine diatoms are considered good candidates for the large-scale cultivation. Among physical parameters, light plays a primary role. Even if sunlight is cost-effective, the employment of artificial light becomes a winning strategy if a high-value microalgal biomass is produced. Several researches on marine diatoms are designed to study the influence of different light regimens to increase biomass production enriched in biotechnologically high-value compounds (lipids, carotenoids, proteins, polysaccharides), or with emphasised photonic properties of the frustule.

Characterization of biodegradation in a 17th century easel painting and potential for a biological approach.

It is important to characterize the microorganisms involved in biodeterioration processes to understand their effects on cultural assets and to define an efficient strategy for protecting artworks, monuments, and buildings from microbiological recolonization. In this study, we analyzed the microbial communities dwelling on the verso (front) and recto (back) sides of a 17th century easel painting attributed to Carlo Bononi, an Italian artist of the first Baroque period. Cultivable bacteria and fungi colonizing the painting were isolated and identified in order to characterize the microbial community possibly involved in deteriorating the pictorial layer of the painting. The isolated bacterial strains belonged to the Staphylococcus and Bacillus genera. Furthermore, culture-dependent techniques and SEM/EDS analyses revealed the presence of filamentous fungi of the genera Aspergillus, Penicillium, Cladosporium, and Alternaria. The chemical compositions of pigments were consistent with typical 17th century paintings, and some of the identified pigments, namely red lac and red and yellow earths, could be exploited as nutrient sources by painting-associated microorganisms. The study also evaluated, in vitro, the potential decontaminating activity of a biocompound, containing spores of Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium. The results indicated the ability of this biocompound to counteract the growth of contaminating microorganisms that are potentially dangerous to the painting, suggesting the potential use of these microorganisms to prevent biodeterioration of artworks.

Enhanced photosynthetic linear electron flow in mixotrophic green microalga Ettlia oleoabundans UTEX 1185.

Basic understanding of the photosynthetic physiology of the oleaginous green microalga Ettlia oleoabundans is still very limited, including the modulation of the photosynthetic membrane upon metabolism conversion from autotrophy to mixotrophy. It was previously reported that, upon glucose supply in the culture medium, E. oleoabundans preserves photosystem II (PSII) from degradation by virtue of a higher packing of thylakoid complexes. In this work, it was investigated whether in the mixotrophic exponential growth phase the PSII activity is merely preserved or even enhanced. Modulated fluorescence parameters were then recorded under short-term treatments with increasing irradiance values of white light. It was found that the mixotrophic microalga down-regulated the chlororespiratory electron recycling from photosystem I (PSI), but enhanced the linear electron flow from PSII to PSI. Ability to keep PSII more open than in autotrophic growth conditions indicated that the respiration of the glucose taken up from the medium fed the carbon fixing reactions with CO2. The overall electron poise was indeed well regulated, with a lesser need for thermal dissipation of excess absorbed energy. It is proposed that the significant, though small, increase in PSII maximum quantum yield in mixotrophic cells just reflects an improved light energy use and an increased photochemical capacity as compared to the autotrophic cells.

In pea stipules a functional photosynthetic electron flow occurs despite a reduced dynamicity of LHCII association with photosystems.

The flexible association of the light harvesting complex II (LHCII) to photosystem (PS) I and PSII to balance their excitation is a major short-term acclimation process of the thylakoid membrane, together with the thermal dissipation of excess absorbed energy, reflected in non-photochemical quenching of chlorophyll fluorescence (NPQ). In Pisum sativum, the leaf includes two main photosynthetic parts, the basal stipules and the leaflets. Since the stipules are less efficient in carbon fixation than leaflets, the adjustments of the thylakoid system, which safeguard the photosynthetic membrane against photodamage, were analysed. As compared to leaflets, the stipules experienced a decay in PSII photochemical activity. The supramolecular organization of photosystems in stipules showed a more conspicuous accumulation of large PSII-LHCII supercomplexes in the grana, but also a tendency to retain the PSI-LHCI-LHCII state transition complex and the PSI-LHCI-PSII-LHCII megacomplexes probably located at the interface between appressed and stroma-exposed membranes. As a consequence, stipules had a lower capacity to perform state transitions and the overall thylakoid architecture was less structurally flexible and ordered than in leaflets. Yet, stipules proved to be quite efficient in regulating the redox state of the electron transport chain and more capable of inducing NPQ than leaflets. It is proposed that, in spite of a relatively static thylakoid arrangement, LHCII interaction with both photosystems in megacomplexes can contribute to a regulated electron flow.

Effect of different CO2 concentrations on biomass, pigment content, and lipid production of the marine diatom Thalassiosira pseudonana.

The marine diatom Thalassiosira pseudonana grown under air (0.04% CO2) and 1 and 5% CO2 concentrations was evaluated to determine its potential for CO2 mitigation coupled with biodiesel production. Results indicated that the diatom cultures grown at 1 and 5% CO2 showed higher growth rates (1.14 and 1.29 div day-1, respectively) and biomass productivities (44 and 48 mgAFDWL-1 day-1) than air grown cultures (with 1.13 div day-1 and 26 mgAFDWL-1 day-1). The increase of CO2 resulted in higher cell volume and pigment content per cell of T. pseudonana. Interestingly, lipid content doubled when air was enriched with 1-5% CO2. Moreover, the analysis of the fatty acid composition of T. pseudonana revealed the predominance of monounsaturated acids (palmitoleic-16:1 and oleic-18:1) and a decrease of the saturated myristic acid-14:0 and polyunsaturated fatty acids under high CO2 levels. These results suggested that T. pseudonana seems to be an ideal candidate for biodiesel production using flue gases.

Frequently asked questions about chlorophyll fluorescence, the sequel.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V /F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

Light acclimation in the lycophyte Selaginella martensii depends on changes in the amount of photosystems and on the flexibility of the light-harvesting complex II antenna association with both photosystems.

Vascular plants have evolved a long-term light acclimation strategy primarily relying on the regulation of the relative amounts of light-harvesting complex II (LHCII) and of the two photosystems, photosystem I (PSI) and photosystem II (PSII). We investigated whether such a model is also valid in Selaginella martensii, a species belonging to the early diverging group of lycophytes. Selaginella martensii plants were acclimated to three natural light regimes (extremely low light (L), medium light (M) and full sunlight (H)) and thylakoid organization was characterized combining ultrastructural, biochemical and functional methods. From L to H plants, thylakoid architecture was rearranged from (pseudo)lamellar to predominantly granal, the PSII : PSI ratio changed in favour of PSI, and the photochemical capacity increased. However, regulation of light harvesting did not occur through variations in the amount of free LHCII, but rather resulted from the flexibility of the association of free LHCII with PSII and PSI. In lycophytes, the free interspersed LHCII serves a fixed proportion of reaction centres, either PSII or PSI, and the regulation of PSI-LHCII(-PSII) megacomplexes is an integral part of long-term acclimation. Free LHCII ensures photoprotection of PSII, allows regulated use of PSI as an energy quencher, and can also quench endangered PSI.

Re-cultivation of Neochloris oleoabundans in exhausted autotrophic and mixotrophic media: the potential role of polyamines and free fatty acids.

Neochloris oleoabundans (Chlorophyta) is widely considered one of the most promising microalgae for biotechnological applications. However, the large-scale production of microalgae requires large amounts of water. In this perspective, the possibility of using exhausted growth media for the re-cultivation of N. oleoabundans was investigated in order to simultaneously make the cultivation more economically feasible and environmentally sustainable. Experiments were performed by testing the following media: autotrophic exhausted medium (E+) and mixotrophic exhausted medium after cultivation with glucose (EG+) of N. oleoabundans cells grown in a 20-L photobioreactor (PBR). Both exhausted media were replenished with the same amounts of nitrate and phosphate as the control brackish medium (C). Growth kinetics, nitrate and phosphate consumption, photosynthetic pigments content, photosynthetic efficiency, cell morphology, and lipid production were evaluated. Moreover, the free fatty acid (FFA) composition of exhausted media and the polyamine (PA) concentrations of both algae and media were analyzed in order to test if some molecules, released into the medium, could influence algal growth and metabolism. Results showed that N. oleoabundans can efficiently grow in both exhausted media, if appropriately replenished with the main nutrients (E+ and EG+), especially in E+ and to the same extent as in C medium. Growth promotion of N. oleoabundans was attributed to PAs and alteration of the photosynthetic apparatus to FFAs. Taken together, results show that recycling growth medium is a suitable solution to obtain good N. oleoabundans biomass concentrations, while providing a more sustainable ecological impact on water resources.