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Sustained H2 photoproduction by green alga Chlamydomonas reinhardtii is achieved under pulse-illumination superimposed on continuous low background light. Pulse-illuminated algae act as biocatalysts producing H2via direct water biophotolysis.

Present agricultural production typically depends on the intensive use of synthetic pesticides with potentially harmful consequences for humans and the environment. To ensure food security for the rapidly rising global population it is necessary to develop more sustainable alternatives to synthetic pesticides. Microalgae possess a large diversity in antimicrobial compounds and are considered one of the most promising sustainable sources of novel biopesticides. Antimicrobial activities of 15 microalgae strains were investigated against a selection of seven common plant pathogens relevant to agricultural production. Several microalgae were identified to possess antimicrobial activity with an extract of Chlorella sorokiniana showing the strongest growth inhibition of the plant pathogen Phytophthora cactorum. Different pre-treatments like freeze-drying, solvents with different polarities, and extraction methods were analyzed in regards to the level of antimicrobial activity of C. sorokiniana. The best C. sorokiniana extract demonstrated potential for biopesticide application on strawberry leaves infected with P. cactorum. This study reveals the abundant potential of microalgae as natural biopesticide for organic or more sustainable regular agriculture.

International audience; Baeyer−Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMO Xeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMO Acineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g DCW −1 at an optical density of OD 750 = 10) and an initial rate of 3.7 ± 0.2 mM h −1. While the cells containing CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h −1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygendependent enzymatic processes.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O

Background Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness. Results A 1.7-fold increase of activity (150 µmol min−1 gDCW−1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L−1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions. Conclusions Results show that under photoautotrophic conditions at a specific activity of 90 µmol min−1 gDCW−1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.

Cell immobilization is a promising approach to create efficient photosynthetic cell factories for sustainable chemical production. Here, we demonstrate a novel photosynthetic solid-state cell factory design for sustainable biocatalytic ethylene production. We entrapped cyanobacteria within never-dried hydrogel films of TEMPO-oxidized cellulose nanofibers (TCNF) cross-linked with polyvinyl alcohol (PVA) to create a self-standing matrix architecture. The matrix is operational in the challenging submerged conditions and outperforms existing alginate-based solutions in terms of wet strength, long-term cell fitness, and stability. Based on rheological investigations, the critical strength of wet TCNF matrices is three times higher than in the existing immobilization matrices of alginate cross-linked with Ca2+. This is due to the rigid nature of the colloidal nanofiber network and the strong cross-linking with PVA, as opposed to polymeric alginate with reversible ionic Ca2+bonds. The porous and hygroscopic nanofiber network also shields the cyanobacterial cells from environmental stress, maintaining photosynthetic activity during partial drying of films, and when submerged in the nutrient medium for long-term cultivation. Finally, TCNF matrices allow the ethylene-producingSynechocystissp. PCC 6803 cells to operate in submerged conditions under high inorganic carbon loads (200 mM NaHCO3), where Ca2+-alginate matrices fail. The latter show severe cell leakage due to matrix disintegration already within 20 min of NaHCO3supplementation. In contrast, TCNF-based matrices prevent cell leakage to the medium and restrict culture growth, leading to improved ethylene production yields. Furthermore, the operational capacity of the self-standing TNCF cell factory can be maintained long-term by periodically refreshing the nutrient medium. All in all, the results showcase the versatility and potential of cell immobilization with the never-dried colloidal TCNF matrix, paving the way towards novel biotechnological pathways using solid-state cell factories designed for efficient and sustainable production ofe.g., monomers and fuels.

Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethylene now used for commercial applications is produced exclusively from non-renewable petroleum sources, while competitive biotechnological production systems do not yet exist. This review focuses on the currently developed photoautotrophic bioproduction strategies that enable direct solar-driven conversion of CO2 into ethylene, based on the use of genetically engineered photosynthetic cyanobacteria expressing heterologous ethylene forming enzyme (EFE) from Pseudomonas syringae. The emphasis is on the different engineering strategies to express EFE and to direct the cellular carbon flux towards the primary metabolite 2-oxoglutarate, highlighting associated metabolic constraints, and technical considerations on cultivation strategies and conditional parameters. While the research field has progressed towards more robust strains with better production profiles, and deeper understanding of the associated metabolic limitations, it is clear that there is room for significant improvement to reach industrial relevance. At the same time, existing information and the development of synthetic biology tools for engineering cyanobacteria open new possibilities for improving the prospects for the sustainable production of renewable ethylene. This article is protected by copyright. All rights reserved.

In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O(2)to H2O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. InSynechocystissp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types have been characterized in cyanobacteria: NDH-1(1)and NDH-1(2), which function in respiration; and NDH-1(3)and NDH-1(4), which function in CO(2)uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants ( increment flv1and Delta flv3) and the double NDH-1 mutants ( increment d1d2, which is deficient in NDH-1(1,2)and increment d3d4, which is deficient in NDH-1(3,4)), we studied triple mutants lacking one of Flv1 or Flv3, and NDH-1(1,2)or NDH-1(3,4). We show that the presence of either Flv1/3 or NDH-1(1,2), but not NDH-1(3,4), is indispensable for survival during changes in growth conditions from high CO2/moderate light to low CO2/high light. Our results show functional redundancy between FDPs and NDH-1(1,2)under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH-1(1,2), allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO(2)and light availability.

Photosynthetic electron transfer comprises a series of light-induced redox reactions catalysed by multiprotein machinery in the thylakoid. These protein complexes possess cofactors susceptible to redox modifications by reactive small molecules. The gaseous radical nitric oxide (NO), a key signalling molecule in green algae and plants, has earlier been shown to bind to Photosystem (PS) II and obstruct electron transfer in plants. The effects of NO on cyanobacterial bioenergetics however, have long remained obscure. In this study, we exposed the model cyanobacterium Synechocystis sp. PCC 6803 to NO under anoxic conditions and followed changes in whole-cell fluorescence and oxidoreduction of P700 in vivo. Our results demonstrate that NO blocks photosynthetic electron transfer in cells by repressing PSII, PSI, and likely the NDH dehydrogenase-like complex 1 (NDH-1). We propose that iron‑sulfur clusters of NDH-1 complex may be affected by NO to such an extent that ferredoxin-derived electron injection to the plastoquinone pool, and thus cyclic electron transfer, may be inhibited. These findings reveal the profound effects of NO on Synechocystis cells and demonstrate the importance of controlled NO homeostasis in cyanobacteria.

Photosynthetic cyanobacteria are exposed to rapid changes in light intensity in their natural habitats, as well as in photobioreactors. To understand the effects of such fluctuations on Synechocystis sp. PCC 6803, the global proteome of cells grown under a fluctuating light condition (low background light interrupted with high light pulses) was compared to the proteome of cells grown under constant light with concomitant acclimation of cells to low CO2 level. The untargeted global proteome of Synechocystis sp. PCC 6803 was analyzed by data-dependent acquisition (DDA), which relies on the high mass accuracy and sensitivity of orbitrap-based tandem mass spectrometry. In addition, a targeted selected reaction monitoring (SRM) approach was applied to monitor the proteomic changes in a strain lacking flavodiiron proteins Flv1 and Flv3. This strain is characterized by impaired growth and photosynthetic activity under fluctuating light. An obvious reprogramming of cell metabolism was observed in this study and was compared to a previous transcriptional analysis performed under the same fluctuating light regime. Cyanobacterial responses to fluctuating light correlated at mRNA and protein levels to some extent, but discrepancies indicate that several proteins are post-transcriptionally regulated (affecting observed protein abundances). The data suggest that Synechocystis sp. PCC 6803 maintain higher nitrogen assimilation, serving as an electron valve, for long-term acclimation to fluctuating light upon CO2 step-down. Although Flv1 and Flv3 are known to be crucial for the cells at the onset of illumination, the flavodiiron proteins, as well as components of carbon assimilation pathways, were less abundant under fluctuating light.

The ability to capture and convert sunlight, water and nutrients into useful compounds make photosynthetic microbes ideal candidates for the bio-industrial factories of the future. However, the suitability of isolates from temperate regions to grow under Nordic conditions is questionable. In this work, we explore the chemotaxonomy of Nordic strains of cyanobacteria and one green alga and evaluate their potential as raw materials for the production of lipid-based bio-industrial compounds. Thin-layer chromatography was used to identify the presence of triacylglycerol, which were detected in the majority of strains. Fatty acid methyl ester profiles were analysed to determine the suitability of strains for the production of biodiesel or the production of polyunsaturated fatty acids for the nutraceutical industry. The Nordic Synechococcus strains were unique in demonstrating fatty acid profiles comprised mostly C14:0, C16:0 and C16:1 and lacking polyunsaturated fatty acids. These properties translated to superior predicted biodiesel qualities, including cetane number, cold filter plugging point and oxidative stability compared to the other evaluated strains. Polyunsaturated fatty acids were detected at high levels (38-53%), with Calothrix sp. 336/3 being abundant in two essential fatty acids, linoleic and alpha-linolenic acid (21 and 17%, respectively). Gamma-linoleic acid was the predominant polyunsaturated fatty acid for the remaining strains (13-21%). In addition to assessing the potential of Nordic strains for bio-industrial production, this work also discusses issues such as taxonomy and predictive modelling, which can affect the identification of prospective high-performing strains.

openaire: EC/H2020/742829/EU//DRIVEN In the effort toward sustainable advanced functional materials, nanocelluloses have attracted extensive recent attention. Nanocelluloses range from rod-like highly crystalline cellulose nanocrystals to longer and more entangled cellulose nanofibers, earlier denoted also as microfibrillated celluloses and bacterial cellulose. In recent years, they have spurred research toward a wide range of applications, ranging from nanocomposites, viscosity modifiers, films, barrier layers, fibers, structural color, gels, aerogels and foams, and energy applications, until filtering membranes, to name a few. Still, nanocelluloses continue to show surprisingly high challenges to master their interactions and tailorability to allow well-controlled assemblies for functional materials. Rather than trying to review the already extensive nanocellulose literature at large, here selected aspects of the recent progress are the focus. Water interactions, which are central for processing for the functional properties, are discussed first. Then advanced hybrid gels toward (multi)stimuli responses, shape-memory materials, self-healing, adhesion and gluing, biological scaffolding, and forensic applications are discussed. Finally, composite fibers are discussed, as well as nanocellulose as a strategy for improvement of photosynthesis-based chemicals production. In summary, selected perspectives toward new directions for sustainable high-tech functional materials science based on nanocelluloses are described.

The growth of the world's population increases the demand for fresh water, food, energy, and technology, which in turn leads to increasing amount of wastewater, produced both by domestic and industrial sources. These different wastewaters contain a wide variety of organic and inorganic compounds which can cause tremendous environmental problems if released untreated. Traditional treatment systems are usually expensive, energy demanding and are often still incapable of solving all challenges presented by the produced wastewaters. Microalgae are promising candidates for wastewater reclamation as they are capable of reducing the amount of nitrogen and phosphate as well as other toxic compounds including heavy metals or pharmaceuticals. Compared to the traditional systems, photosynthetic microalgae require less energy input since they use sunlight as their energy source, and at the same time lower the carbon footprint of the overall reclamation process. This mini-review focuses on recent advances in wastewater reclamation using microalgae. The most common microalgal strains used for this purpose are described as well as the challenges of using wastewater from different origins. We also describe the impact of climate with a particular focus on a Nordic climate. Special Issue

NordAqua is a multidisciplinary Nordic Center of Excellence funded by NordForsk Bioeconomy program (2017–2022). The research center promotes Blue Bioeconomy and endeavours to reform the use of natural resources in a environmentally sustainable way. In this short communication, we summarize particular outcomes of the consortium. The key research progress of NordAqua includes (1) improving of photosynthetisis, (2) developing novel photosynthetic cell factories that function in a “solar-driven direct CO2 capture to target bioproducts” mode, (3) promoting the diversity of Nordic cyanobacteria and algae as an abundant and resilient alternative for less sustainable forest biomass and for innovative production of biochemicals, and (4) improving the bio-based wastewater purification and nutrient recycling technologies to provide new tools for integrative circular economy platforms.

Significance Photosynthetic H2 production in the green alga Chlamydomonas reinhardtii is catalyzed by O2-sensitive [FeFe]-hydrogenases, which accept electrons from photosynthetically reduced ferredoxin and reduce protons to H2. Since the process occurs downstream of photosystem I, the contribution of photosystem II (PSII) in H2 photoproduction has long been a subject of debate. Indeed, water oxidation by PSII results in O2 accumulation in chloroplasts, which inhibits H2 evolution. Therefore, clear evidence for direct water biophotolysis resulting in simultaneous H2 and O2 releases in algae has never been presented. This paper demonstrates that sustained H2 photoproduction in C. reinhardtii is directly linked to PSII-dependent water oxidation and brings insights into regulation of PSII activity and H2 production by CO2/HCO3– under microoxic conditions., The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and H218O, we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h-1 L-1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

Photomixotrophy is a metabolic state that enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we demonstrate, via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual inhibition of Q(A)(−) reoxidation in wild-type Synechocystis, which largely decreases photosynthesis over 3 d of growth. This process is circumvented by deleting the gene encoding cytochrome c(M) (CytM), a cryptic c-type heme protein widespread in cyanobacteria. The ΔCytM strain maintained active photosynthesis over the 3-d period, demonstrated by high photosynthetic O(2) and CO(2) fluxes and effective yields of PSI and PSII. Overall, this resulted in a higher growth rate compared to that of the wild type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localized respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a phenotype similar to that of ΔCytM under photomixotrophy. This, in combination with other physiological data, and in contrast to a previous hypothesis, suggests that CytM does not transfer electrons to these complexes. In summary, our data suggest that CytM may have a regulatory role in photomixotrophy by modulating the photosynthetic capacity of cells.

In oxygenic photosynthetic organisms excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2 to H2O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types haven been characterized in cyanobacteria: NDH-11 and NDH-12, which function in respiration; and NDH-13 and NDH-14, which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (Δflv1 and Δflv3) and the double NDH-1 mutants (Δd1d2, which is deficient in NDH-11,2 and Δd3d4, which is deficient in NDH-13,4), we studied triple mutants lacking either one of Flv1 or Flv3, and NDH-11,2 or NDH-13,4. We show that the presence of either Flv1/3 or NDH-11,2, but not NDH-13,4, is indispensable for survival during changes in growth conditions from high CO2 /moderate light to low CO2 / high light. Our results suggest functional redundancy and crosstalk between FDPs and NDH-11,2 under the studied conditions, and demonstrate that the functions of FDPs and NDH-11,2 are dynamically coordinated for the efficient oxidation of PSI and for photoprotection under variable CO2 and light availability.One sentence summaryFlavodiiron proteins and NDH-1 complex ensure survival of cyanobacterial cells by cooperatively safeguarding the photosynthetic apparatus against excessive reduction

A novel thin-layer biocatalyst for photosynthetic N2 fixation and H2 photoproduction was assembled using a Ca2+-alginate matrix and heterocysts isolated from wild-type Anabaena sp. PCC 7120 filaments. Compared to suspension heterocysts, heterocysts entrapped in Ca2+-alginate films showed improved stability of the nitrogenase system. While suspension heterocysts lost nitrogenase activity within 24 h, immobilized heterocysts supported nitrogenase activity for up to 125 h. The maximum specific rate of acetylene reduction was the same in both cases (∼0.4 μmol C2H2 mg Chl−1 h−1), but the catalyst with entrapped heterocysts required a much longer time to achieve the maximum rate (60 h instead of 3 h in suspension). Simultaneously with acetylene reduction, the immobilized heterocysts were able to photoproduce H2 for 125 h, yielding up to 1.1 mmol H2 mg Chl−1. The absence of acetylene increased the H2 photoproduction rate to a maximum of 25–30 μmol H2 mg Chl−1 h−1, and the catalyst was capable of H2 photoproduction for 190 h, yielding up to 2.5 mmol H2 mg Chl−1. The recovery of the catalyst with entrapped heterocysts was achieved through placing the cells in a N2 atmosphere for 24 h. This engaged a second cycle of H2 photoproduction, which lasted for another 240 h (10 days), thus yielding ∼3 mmol H2 mg Chl−1 in total after 454 h. Together, these findings demonstrate great potential for a heterocyst-based thin-layer platform for the sustainable production of chemicals and biofuels.

Cyanobacteria and green algae harness solar energy to split water and to fix CO 2 . Under specific conditions, they are capable of photoproduction of molecular hydrogen (H 2 ). This study compares the light-energy-to-hydrogen-energy conversion efficiency (LHCE) in two heterocystous, N 2 -fixing cyanobacteria (wild-type Calothrix sp. strain 336/3 and the Δ hupL mutant of Anabaena sp. strain PCC 7120) and in the sulfur-deprived green alga, Chlamydomonas reinhardtii strain CC-124, after entrapment of the cells in thin Ca 2+ -alginate films. The experiments, performed under photoautotrophic conditions, showed higher LHCEs in the cyanobacteria as compared to the green alga. The highest efficiency of ca. 2.5% was obtained in films of the entrapped Δ hupL strain under low light condition (2.9 W m −2 ). Calothrix sp. 336/3 films produced H 2 with a maximum efficiency of 0.6% under 2.9 W m −2 , while C. reinhardtii films produced H 2 most efficiently under moderate light (0.14% at 12.1 W m −2 ). Exposure of the films to light above 16 W m −2 led to noticeable oxidative stress in all three strains, which increased with light intensity. The presence of oxidative stress was confirmed by increased ( i ) degradation of chlorophylls and some structural carotenoids (such as β -carotene), ( ii ) production of hydroxylated carotenoids (such as zeaxanthin), and ( iii ) carbonylation of proteins. We conclude that the H 2 photoproduction efficiency in immobilized algae and cyanobacteria can be further improved by entrapping cultures in immobilization matrices with increased permeability for gases, especially oxygen, while matrices with low porosity produced increased amounts of xanthophylls and other antioxidant compounds.

NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well-characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (ΔndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the ΔndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth conditions.

Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.

In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the sll0218 and sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting. This work clarifies and dissects the roles of the flv4-2 operon-encoded proteins, Flv2/Flv4 heterodimer and the elusive Sll0218, in photoprotection of the photosynthetic apparatus in Synechosystis. While Flv2/Flv4 heterodimer is involved in an alternative electron transfer route, the Sll0218 protein is localized to specific cell compartments where photosynthetic complexes are assembled, and it is involved in the stabilization of Photosystem II complexes.

Photomixotrophy is a metabolic state, which enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we show via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual reduction of the plastoquinone pool in wild-type Synechocystis, which fully downscales photosynthesis over three days of growth. This process is circumvented by deleting the gene encoding cytochrome cM (CytM), a cryptic c-type heme protein widespread in cyanobacteria. ΔCytM maintained active photosynthesis over the three day period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of Photosystem II and Photosystem I. Overall, this resulted in a higher growth rate than wild-type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a similar phenotype under photomixotrophy to ΔCytM, demonstrating that CytM is not transferring electrons to these complexes, which has previously been suggested. In summary, the obtained data suggests that CytM may have a regulatory role in photomixotrophy by reducing the photosynthetic capacity of cells.One sentence summaryThe cryptic, highly conserved cytochrome cM completely blocks photosynthesis in Synechocystis under three days of photomixotrophy, possibly by suppressing CO2 assimilation.

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in all life Domains. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4) essential for photoprotection of photosynthesis. A direct comparison of the Mehler-like reaction (O2 photoreduction) in high Ci (3% CO2, HC) and low Ci (air level CO2, LC) acclimated cells demonstrated that the Flv1/Flv3 heterodimer is responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions Flv1/Flv3 acts only as a transient electron sink due to competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

Additional file 1: Figure S1. Schematic experimental setup of short-term pulse illumination experiments. In total, 19 ml of C. reinhardtii wt and flv mutant cultures grown in TP medium for 2 days in 50 µmol photons m−2 s−1 bubbling with 3% CO2 was withdrawn and subjected to less than 10-min darkness together with Ar purging before the 20-min pulse illumination experiments. Figure S2. Short-term H2 photoproduction under 1-s light/9-s dark pulse illumination protocol in two different knockout flv mutant lines. Cells were grown for 2 days at 50 µmol photons m−2 s−1 in TP medium bubbling with 3% CO2, transferred to a vial equipped with H2 and O2 sensors, flushed with Ar. The intensity of light pulses was around 250 µmol photons m−2 s−1. H2 yields during 10-min dark anaerobic adaptation phase, 20-min H2 photoproduction phase and 3-min dark H2 uptake phase in CC-4533, flv 208 and flv 791. Experiments have been performed in 3 independent replicates and are presented exemplary. Figure S3. Photosynthetic characteristics of C. reinhardtii wt and flv 208 mutant cultures grown in TP medium for 4 days in 50 µmol photons m−2 s−1 bubbling with 3% CO2. (a) Chl concentration (mg L−1) over a growth period of 4 days. The arrows show when the cultures were withdrawn for further treatments in the long- or short-term experiments. (b) Maximum quantum efficiency of PSII (FV/FM), (c) dark respiration and (d) effective yield of PSII (Y(II)) during 3 days of growth. Experiments have been performed in 5 independent replicates (± SD). (b, d) No statistical significance between wt CC-4533 and flv 208. (c) Statistical significance levels: *p

To guarantee year-round horticultural production, Nordic countries rely on greenhouses to cope with the harsh winter conditions. Despite substantial progress in greenhouse operation, greenhouse effluents are still overloaded with nutrients and are known to be a source of eutrophication that decreases the quality of natural waters worldwide. Here we investigate the possibility of recirculating hydroponic effluents from a commercial cucumber greenhouse to cultivate photoautotrophic microalgal biomass. A stepwise methodology was applied to scale-up procedures from laboratory to a pilot scale photobioreactor (PBR) which was operated inside a greenhouse. The screening results identified that 12 (of 13) strains were capable of proliferating in the hydroponic effluent without any adjustment or dilution. A Nordic microalga from the Scenedesmaceae family was selected for a PBR operation which lasted 36 days. This continuous cultivation was undertaken under four different test conditions and removal efficiencies between 18 and 35% for N-NO3− and 40–98% for P-PO43− were achieved. Over 1000 L of hydroponic effluent was recirculated, with a steady N-NO3− uptake of 86–92 mg g−1 and a P-PO43− uptake of 11–21 mg g−1 biomass. These results demonstrated that a hydroponic effluent N:P of 15–26 and pH of 7.5 were the most appropriate conditions for nutrient uptake, although increasing the pH to 9 may promote P-PO43− uptake. Overall, this work indicates the feasibility of microalgal bioremediation of greenhouse effluents in the Nordic countries.

The carotenoid composition of the filamentous heterocystous N2-fixing cyanobacterium Calothrix sp. 336/3 was investigated under three conditions: in full medium (non-diazotrophic growth); in the absence of combined nitrogen (diazotrophic growth); and after long-term H2 photoproduction (diazotrophic medium and absence of nitrogen in the atmosphere). Anabaena sp. PCC 7120 and its ΔhupL mutant with disrupted uptake hydrogenase were used as reference strains. Analysis of identified carotenoids and enzymes involved in carotenogenesis showed the presence of three distinct biosynthetic pathways in Calothrix sp. 336/3. The first one is directed towards biosynthesis of myxoxanthophylls, such as myxol 2'-methylpentoside and 2-hydroxymyxol 2'-methylpentoside. The second pathway results in production of hydroxylated carotenoids, such as zeaxanthin, caloxanthin and nostoxanthin, and the last pathway is responsible for biosynthesis of echinenone and hydroxylated forms of ketocarotenoids, such as 3'-hydroxyechinenone and adonixanthin. We found that carotenogenesis in filamentous heterocystous cyanobacteria varies depending on the nitrogen status of the cultures, with significant accumulation of echinenone during diazotrophic growth at the expense of β-carotene. Under the severe N deficiency and high CO2 supply, which leads to efficient H2 photoproduction, cyanobacteria degrade echinenone and β-carotene, and accumulate glycosylated and hydroxylated carotenoids, such as myxol (or ketomyxol) 2'-methylpentosides, 3'-hydroxyechinenone and zeaxanthin. We suggest that the stability of the photosynthetic apparatus in Calothrix sp. 336/3 cells under N deficiency and high carbon conditions, which also appeared as the partial recovery of the pigment composition by the end of the long-term (∼1 month) H2 photoproduction process, might be mediated by a high content of hydroxycarotenoids.

The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3. By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2. Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT.

Photosynthetic organisms have evolved numerous photoprotective mechanisms and alternative electron sinks/pathways to fine-tune the photosynthetic apparatus under dynamic environmental conditions, such as varying carbon supply or fluctuations in light intensity. In cyanobacteria flavodiiron proteins (FDPs) protect the photosynthetic apparatus from photodamage under fluctuating light (FL). In Arabidopsis thaliana, which does not possess FDPs, the PGR5-related pathway enables FL photoprotection. The direct comparison of the pgr5, pgrl1 and flv knockout mutants of Chlamydomonas reinhardtii grown under ambient air demonstrates that all three proteins contribute to the survival of cells under FL, but to varying extents. The FDPs are crucial in providing a rapid electron sink, with flv mutant lines unable to survive even mild FL conditions. In contrast, the PGRL1 and PGR5-related pathways operate over relatively slower and longer time-scales. Whilst deletion of PGR5 inhibits growth under mild FL, the pgrl1 mutant line is only impacted under severe FL conditions. This suggests distinct roles, yet a close relationship, between the function of PGR5, PGRL1 and FDP proteins in photoprotection.

With the tremendous progress of the past decades, molecular plant science is becoming more unified than ever. We now have the exciting opportunity to further connect subdisciplines and understand plants as whole organisms, as will be required to efficiently utilize them in natural and agricultural systems to meet human needs. The subfields of photosynthesis, plant developmental biology and plant stress are used as examples to discuss how plant science can become better integrated. The challenges, strategies and rich opportunities for the integration of the plant sciences are discussed. In recent years, more and more overlap between various subdisciplines has been inadvertently discovered including tradeoffs that may occur in plants engineered for biotechnological applications. Already important, bioinformatics and computational modelling will become even more central to structuring and understanding the ever growing amounts of data. The process of integrating and overlapping fields in plant biology research is advancing, but plant science will benefit from dedicating more effort and urgency to reach across its boundaries.

The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O(2) in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O(2) downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO(2) levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H(2) photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport.

Flavodiiron proteins (FDPs, also called flavoproteins, Flvs) are modular enzymes widely present in Bacteria and Archaea. The evolution of cyanobacteria and oxygenic photosynthesis occurred in concert with the modulation of typical bacterial FDPs. Present cyanobacterial FDPs are composed of three domains, the β-lactamase-like, flavodoxin-like and flavin-reductase like domains. Cyanobacterial FDPs function as hetero- and homodimers and are involved in the regulation of photosynthetic electron transport. Whilst Flv2 and Flv4 proteins are limited to specific cyanobacterial species (β-cyanobacteria) and function in photoprotection of Photosystem II, Flv1 and Flv3 proteins, functioning in the “Mehler-like” reaction and safeguarding Photosystem I under fluctuating light conditions, occur in nearly all cyanobacteria and additionally in green algae, mosses and lycophytes. Filamentous cyanobacteria have additional FDPs in heterocyst cells, ensuring a microaerobic environment for the function of the nitrogenase enzyme under the light. Here, the evolution, occurrence and functional mechanisms of various FDPs in oxygenic photosynthetic organisms are discussed.

Oxygenic photosynthetic organisms experience strong fluctuations in light intensity in their natural terrestrial and aquatic growth environments. Recent studies with both plants and cyanobacteria have revealed that Photosystem (PS) I is the potential target of damage upon abrupt changes in light intensity. Photosynthetic organisms have, however, developed powerful mechanisms in order to protect their photosynthetic apparatus against such potentially hazardous light conditions. Although the electron transfer chain has remained relatively unchanged in both plant chloroplasts and their cyanobacterial ancestors, the photoprotective and regulatory mechanisms of photosynthetic light reactions have experienced conspicuous evolutionary changes. In cyanobacteria, the specific flavodiiron proteins (Flv1 and Flv3) are responsible for safeguarding PSI under rapidly fluctuating light intensities, whilst the thylakoid located terminal oxidases are involved in the protection of PSII during 12h diurnal cycles involving abrupt, square-wave, changes from dark to high light. Higher plants such as Arabidopsis thaliana have evolved different protective mechanisms. In particular, the PGR5 protein controls electron flow during sudden changes in light intensity by allowing the regulation mostly via the Cytochrome b6f complex. Besides the function of PGR5, plants have also acquired other dynamic regulatory mechanisms, among them the STN7-related LHCII protein phosphorylation that is similarly responsible for protection against rapid changes in the light environment. The green alga Chlamydomonas reinhardtii, as an evolutionary intermediate between cyanobacteria and higher plants, probably possesses both protective mechanisms. In this review, evolutionarily different photoprotective mechanisms under fluctuating light conditions are described and their contributions to cyanobacterial and plant photosynthesis are discussed.

Oxygenic photosynthesis crucially depends on proteins that possess Fe (2+) or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe (2+)-containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms. German Federal Ministry of Education and Research [0316165] DFG [HE 2544/9-1] Academy of Finland [253269, 271832, 273870] Portuguese Fundacao para a Ciencia e a Tecnologia [IF/00881/2013, UID/Multi/04326/2013-CCMAR] European Commission FP7 Marie Curie Initial Training Network "Photo.COMM'' [317184] info:eu-repo/semantics/publishedVersion