Marine microalgae for outdoor biomass production-A laboratory study simulating seasonal light and temperature for the west coast of Sweden.

At Nordic latitudes, year-round outdoor cultivation of microalgae is debatable due to seasonal variations in productivity. Shall the same species/strains be used throughout the year, or shall seasonal-adapted ones be used? To elucidate this, a laboratory study was performed where two out of 167 marine microalgal strains were selected for intended cultivation at the west coast of Sweden. The two local strains belong to Nannochloropsis granulata (Ng) and Skeletonema marinoi (Sm142). They were cultivated in photobioreactors and compared in conditions simulating variations in light and temperature of a year divided into three growth seasons (spring, summer and winter). The strains grew similarly well in summer (and also in spring), but Ng produced more biomass (0.225 vs. 0.066 g DW L-1 day-1 ) which was more energy rich (25.0 vs. 16.6 MJ kg-1 DW). In winter, Sm142 grew faster and produced more biomass (0.017 vs. 0.007 g DW L-1 day-1 ), having similar energy to the other seasons. The higher energy of the Ng biomass is attributed to a higher lipid content (40 vs. 16% in summer). The biomass of both strains was richest in proteins (65%) in spring. In all seasons, Sm142 was more effective in removing phosphorus from the cultivation medium (6.58 vs. 4.14 mg L-1 day-1 in summer), whereas Ng was more effective in removing nitrogen only in summer (55.0 vs. 30.8 mg L-1 day-1 ). Our results suggest that, depending on the purpose, either the same or different local species can be cultivated, and are relevant when designing outdoor studies.

K+ and Cl- channels/transporters independently fine-tune photosynthesis in plants.

In variable light environments, plants adjust light use in photosynthetic electron transport and photoprotective dissipation in the thylakoid membrane. In this respect, roles of the K+/H+ antiporter KEA3, the Cl- channel/transporter CLCe and the voltage-dependent Cl- channel VCCN1 have been unraveled in Arabidopsis thaliana. Here we report that they independently adjust photosynthesis on the basis of analyses using single and higher order loss-of-function mutants. In short experiments of photosynthetic response on transition from dark to low light, we reveal a sequential functioning of VCCN1 and CLCe in the activation of photoprotection and of KEA3 in its downregulation to a low steady state while adjusting the electron transport. On transition from low to high light, VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. Based on parallel electrochromic band shift measurements, the mechanism behind is that VCCN1 builds up a pH gradient across the thylakoid membrane, whereas KEA3 dissipates this gradient, which affects photoprotection. CLCe regulates photosynthesis by a pH-independent mechanism likely involving Cl- homeostasis. Nevertheless, all genotypes grow well in alternating high and low light. Taken together, the three studied ion channels/transporters function independently in adjusting photosynthesis to the light environment.

Functional Expression of Gloeobacter Rhodopsin in PSI-Less Synechocystis sp. PCC6803.

The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the ΔPSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.

Microalgae biotechnology in Nordic countries - the potential of local strains.

Climate change, energy use and food security are the main challenges that our society is facing nowadays. Biofuels and feedstock from microalgae can be part of the solution if high and continuous production is to be ensured. This could be attained in year-round, low cost, outdoor cultivation systems using strains that are not only champion producers of desired compounds but also have robust growth in a dynamic climate. Using microalgae strains adapted to the local conditions may be advantageous particularly in Nordic countries. Here, we review the current status of laboratory and outdoor-scale cultivation in Nordic conditions of local strains for biofuel, high-value compounds and water remediation. Strains suitable for biotechnological purposes were identified from the large and diverse pool represented by saline (NE Atlantic Ocean), brackish (Baltic Sea) and fresh water (lakes and rivers) sources. Energy-efficient annual rotation for cultivation of strains well adapted to Nordic climate has the potential to provide high biomass yields for biotechnological purposes.

Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis: Proteorhodopsin expression increases growth rate and fitness of a ∆PSI strain of Synechocystis sp. PCC6803.

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities.

Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded ß-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α1ß·α2ß-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.

Insights into the Cyanobacterial Deg/HtrA Proteases.

Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.

Regulation of the scp Genes in the Cyanobacterium Synechocystis sp. PCC 6803--What is New?

In the cyanobacterium Synechocystis sp. PCC 6803 there are five genes encoding small CAB-like (SCP) proteins, which have been shown to be up-regulated under stress. Analyses of the promoter sequences of the scp genes revealed the existence of an NtcA binding motif in two scp genes, scpB and scpE. Binding of NtcA, the key transcriptional regulator during nitrogen stress, to the promoter regions was shown by electrophoretic mobility shift assay. The metabolite 2-oxoglutarate did not increase the affinity of NtcA for binding to the promoters of scpB and scpE. A second motif, the HIP1 palindrome 5' GGCGATCGCC 3', was detected in the upstream regions of scpB and scpC. The transcription factor encoded by sll1130 has been suggested to recognize this motif to regulate heat-responsive genes. Our data suggest that HIP1 is not a regulatory element within the scp genes. Further, the presence of the high light regulatory (HLR1) motif was confirmed in scpB-E, in accordance to their induced transcriptions in cells exposed to high light. The HLR1 motif was newly discovered in eight additional genes.

Presence of state transitions in the cryptophyte alga Guillardia theta.

Plants and algae have developed various regulatory mechanisms for optimal delivery of excitation energy to the photosystems even during fluctuating light conditions; these include state transitions as well as non-photochemical quenching. The former process maintains the balance by redistributing antennae excitation between the photosystems, meanwhile the latter by dissipating excessive excitation inside the antennae. In the present study, these mechanisms have been analysed in the cryptophyte alga Guillardia theta. Photoprotective non-photochemical quenching was observed in cultures only after they had entered the stationary growth phase. These cells displayed a diminished overall photosynthetic efficiency, measured as CO2 assimilation rate and electron transport rate. However, in the logarithmic growth phase G. theta cells redistributed excitation energy via a mechanism similar to state transitions. These state transitions were triggered by blue light absorbed by the membrane integrated chlorophyll a/c antennae, and green light absorbed by the lumenal biliproteins was ineffective. It is proposed that state transitions in G. theta are induced by small re-arrangements of the intrinsic antennae proteins, resulting in their coupling/uncoupling to the photosystems in state 1 or state 2, respectively. G. theta therefore represents a chromalveolate algae able to perform state transitions.

Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers.

The serine type Deg/HtrA proteases are distributed in a wide range of organisms from Escherichia coli to humans. The cyanobacterium Synechocystis sp. PCC 6803 possesses three Deg protease orthologues: HtrA, HhoA and HhoB. Previously we compared Synechocystis 6803 wild type cells exposed to mild or severe stress conditions with a mutant lacking all three Deg proteases and demonstrated that stress had strong impact on the proteomes and metabolomes. To identify the biochemical processes, which this protease family is involved in, here we compared Synechocystis sp. PCC 6803 wild type cells with a mutant lacking all three Deg proteases grown under normal growth conditions (30°C and 40 µmol photons m(-2) s(-1)). Deletion of the Deg proteases lead to the down-regulation of proteins related to the biosynthesis of outer cell layers (e.g. the GDP mannose 4,6-dehydratase) and affected protein secretion. During the late growth phase of the culture Deg proteases were found to be secreted to the extracellular medium of the Synechocystis sp. PCC 6803 wild type strain. While cyanobacterial Deg proteases seem to act mainly in the periplasmic space, deletion of the three proteases influences the proteome and metabolome of the whole cell. Impairments in the outer cell layers of the triple mutant might explain the higher sensitivity toward light and oxidative stress, which was observed earlier by Barker and coworkers.

Synergy: a web resource for exploring gene regulation in Synechocystis sp. PCC6803.

Despite being a highly studied model organism, most genes of the cyanobacterium Synechocystis sp. PCC 6803 encode proteins with completely unknown function. To facilitate studies of gene regulation in Synechocystis, we have developed Synergy (http://synergy.plantgenie.org), a web application integrating co-expression networks and regulatory motif analysis. Co-expression networks were inferred from publicly available microarray experiments, while regulatory motifs were identified using a phylogenetic footprinting approach. Automatically discovered motifs were shown to be enriched in the network neighborhoods of regulatory proteins much more often than in the neighborhoods of non-regulatory genes, showing that the data provide a sound starting point for studying gene regulation in Synechocystis. Concordantly, we provide several case studies demonstrating that Synergy can be used to find biologically relevant regulatory mechanisms in Synechocystis. Synergy can be used to interactively perform analyses such as gene/motif search, network visualization and motif/function enrichment. Considering the importance of Synechocystis for photosynthesis and biofuel research, we believe that Synergy will become a valuable resource to the research community.

Co-expression analysis, proteomic and metabolomic study on the impact of a Deg/HtrA protease triple mutant in Synechocystis sp. PCC 6803 exposed to temperature and high light stress.

Members of the DegP/HtrA protease family are widespread in nature and play an important role in proteolysis of misfolded and damaged proteins. The cyanobacterium Synechocystis sp. PCC 6803 contains three Deg proteases, HhoA (Sll1679), HhoB (Sll1427) and HtrA (Slr1204). Using the proteomic or metabolomic approach we investigated a triple deletion mutant (Δdeg) exposed to light or temperature stress. To cope with the stress conditions the triple mutant reduces its energy metabolism and stress-related proteins are induced to protect the cells. Additionally the co-expression of the genes encoding the three proteases with other genes in Synechocystis sp. PCC 6803 was analyzed. While HhoA seems to be involved in house-keeping processes related to protein (re)folding, protein clearance and signaling, the hhoB expression cluster is dominated by genes encoding periplasmic proteins linked to metabolism or signal transduction pathways. The htrA expression pattern is similar to that of genes encoding proteins of the electron transport chain, iron- and nitrogen metabolism. Our integrative approach indicates significant rearrangements in cells depleted of the Deg/HtrA proteases when exposed to stress, both, in the cytoplasmic and extracytoplasmic space.

The search for new chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803.

Light harvesting provides a major challenge in the production of biofuels from microorganisms; while sunlight provides the energy necessary for biomass/biofuel production, at the same time it damages the cells. The genome of Synechocystis sp. PCC 6803 was searched for open reading frames that might code for yet unidentified chlorophyll-binding proteins with low molecular mass that could be involved in stress-adaptation. Amongst 9167 hypothetical ORFs corresponding to potential polypeptides of 100 amino acids or less, two were identified that had the potential to be pigment-binding, because they (i) encoded a potential transmembrane region, (ii) showed sequence similarity with known chlorophyll-binding domains, (iii) were conserved in other cyanobacterial species, and (iv) their codon adaptation index indicated significant translation probability. The two ORFs were located complementary (antisense) and internal to the ferrochelatase (hemH) and the pyruvate dehydrogenase (pdh) genes and therefore were named a-fch and a-pdh, respectively. Transcription of both genes was confirmed; however, no translated proteins could be detected immunologically. Whereas mutations within a-pdh or a-fch did not lead to any obvious phenotype, it is clear that transcripts and proteins over and above the currently known set may play a role in defining the physiology of cyanobacteria and other organisms.

D1 protein turnover is involved in protection of Photosystem II against UV-B induced damage in the cyanobacterium Arthrospira (Spirulina) platensis.

By using two strains of Arthrospira (Spirulina)platensis, an economically important filamentous cyanobacterium, we compared the impairment of PSII activity and loss of D1 protein content under UV-B radiation. Our study showed that UV-B radiation induced a gradual loss of the oxygen-evolving activity to about 56% after 180 min UV-B irradiation both in strains 439 and D-0083, which have been kept under indoor and an outdoor culturing conditions, respectively for a prolonged period of time. The loss of oxygen evolution was accelerated in both strains in the presence of lincomycin, an inhibitor of protein synthesis, and the amount of D1 protein showed a decrease comparable to that of oxygen evolution during the UV-B exposure. However, the UV-B induced loss of oxygen-evolving activity and D1 protein amount was largely prevented when A. platensis cells were exposed to UV-B irradiance supplemented with visible light. Comparison of the two strains also showed a smaller extent of D1 protein synthesis dependent PSII repair in the indoor strain. Our results show that turnover of the D1 protein is an important defense mechanism to counteract the UV-B induced damage of PSII in A. platensis, and also that visible light plays an important role in maintaining the function of PSII under simultaneous exposure to UV-B and visible light.

Differential regulation of psbA and psbD gene expression, and the role of the different D1 protein copies in the cyanobacterium Thermosynechococcus elongatus BP-1.

In Thermosynechococcus elongatus BP-1, which is the preferred organism in recent structural studies of PSII, three psbA and two psbD genes code for three D1 and one D2 protein isoforms, respectively. The regulation and function of these genes and protein products is largely unknown. Therefore, we used quantitative RT-PCR to follow changes in the mRNA level of the respective genes, in combination with biophysical measurements to detect changes in the electron transport activity of Photosystem II under exposure to different visible and UV light, and temperature conditions. In cells which are acclimated to 40 micromol m(-2)s(-1) growth light conditions at 40 degrees C the main populations of the psbA and psbD transcripts arise from the psbA1 and psbD1 genes, respectively. When the temperature is raised to 60 degrees C psbA1 becomes the single dominating psbA mRNA species. Upon exposure of the cells to 500 micromol m(-2)s(-1) intensity visible light psbA3 replaces psbA1 as the dominating psbA mRNA species, and psbD2 increases at the expense of psbD1. UV-B radiation also increases the abundance of psbA3, and psbD2 at the expense of psbA1 and psbD1, respectively. From the different extent of total D1 protein loss in the absence and presence of lincomycin it was estimated that the PsbA3 protein isoform replaces PsbA1 in about 65% of PSII centers after 2 h of high light acclimation. Under the conditions of different psbA transcript distributions chlorophyll fluorescence and thermoluminescence measurements were applied to monitor charge recombination characteristics of the S2Q(A)(-) and S2Q(B)(-) states. We obtained faster decay of flash-induced chlorophyll fluorescence in the presence of DCMU, as well as lower peak temperature of the Q and B thermoluminescence bands when PsbA3 replaced PsbA1 as the main D1 protein isoform. The relevance of dynamic changes in the abundance of psbA and psbD transcript levels, as well as D1 protein isoforms in the acclimation of T. elongatus to changing environmental conditions is discussed.

The psbA gene family responds differentially to light and UVB stress in Gloeobacter violaceus PCC 7421, a deeply divergent cyanobacterium.

Gloeobacter violaceus PCC 7421 is a slow-growing cyanobacterium which lacks thylakoid membranes, but whose five-membered psbA gene family encodes three isoform variants of the PsbA (D1) reaction center protein of Photosystem II. Under standard culture conditions Gloeobacter exhibits photosystem II electron transport, but several clear modifications in the redox potential of key cofactors bound by the PsbA protein are manifested in the flash-fluorescence characteristics. In other cyanobacteria dynamic expression of multiple psbA genes and turnover of PsbA isoforms is critical to counter excitation stress. We found that each of Gloeobacter's five psbA genes is expressed, with transcript abundances spanning 4.5 orders of magnitude. psbAI (glr2322) and psbAII (glr0779), encoding identical PsbA:2 form proteins, are constitutively expressed and dominate the psbA transcript pool under control conditions. psbAIII (gll3144) was strongly induced under photoinhibitory high irradiance stress, thereby contributing to a large increase in the psbA transcript pool that allowed cells to maintain their PsbA protein pools and then recover from irradiance stress, within one cellular generation. In contrast, under comparable photoinhibition provoked by UVB the cells were unable to maintain their psbA transcript and PsbA protein pools, and showed limited subsequent recovery. psbAIV (glr1706) and psbAV (glr2656), encoding two divergent PsbA isoforms, showed consistent trace expression but were never quantitatively significant contributors to the psbA transcript pool.

Molecular mechanisms of light stress of photosynthesis.

Photosynthesis is the basic energy conversion process on Earth, which makes possible the utilization of the energy of sunlight for living organisms. However, light is not only the basic driving force of photosynthesis, but also an important stress factor at the same time. Light-induced decline of photosynthetic activity, generally denoted as photoinhibition, is a general phenomenon in all oxygenic photosynthetic organism under conditions when the metabolic processes cannot keep up with the electron flow produced by the primary photoreactions. Although light-induced damage occurs in all pigmented photosynthetic complexes the primary site of photoinhibition is the photosystem II (PSII) complex, which performs light-driven oxidation of water to protons and oxygen. The main factors, which are responsible for the light sensitivity of photosystem II, are excited pigment molecules, oxygen, manganese, as well as electron donors with high-oxidizing potential. Photosystem II can be efficiently protected from photodamage by the combination of harmless dissipation of absorbed light energy, nonradiative charge recombination, and repair of damaged reaction center complexes, making possible the safe utilization of light, the highly energetic substrate of photosynthesis.

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803.

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.

Cyclic nucleotides, the photosynthetic apparatus and response to a UV-B stress in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyclic nucleotides cAMP and cGMP are ubiquitous signaling molecules that mediate many adaptative responses in eukaryotic cells. Cyanobacteria present the peculiarity among the prokaryotes of having the two types of cyclic nucleotide. Cellular homeostasis requires both cyclases (adenylyl/guanylyl, for their synthesis) and phosphodiesterases (for their degradation). Fully segregated null mutants have been obtained for the two genes, sll1624 and slr2100, which encode putative cNMP phosphodiesterases. We present physiological evidence that the Synechocystis PCC 6803 open reading frame slr2100 could be a cGMP phosphodiesterase. In addition, we show that Slr2100, but not Sll1624, is required for the adaptation of the cells to a UV-B stress. UV-B radiation has deleterious effects for photosynthetic organisms, in particular on the photosystem II, through damaging the protein structure of the reaction center. Using biophysical and biochemical approaches, it was found that Slr2100 is involved in the signal transduction events which permit the repair of the UV-B-damaged photosystem II. This was confirmed by quantitative reverse transcriptase-PCR analyses. Altogether, the data point to an important role for cGMP in signal transduction and photoacclimation processes during a UV-B stress.