Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

TOR kinase activity in Chlamydomonas reinhardtii is modulated by cellular metabolic states.

Target of rapamycin (TOR) kinase is a sensor and a central integrator of internal and external metabolic cues. However, in algae and in higher plants, the components of TOR kinase signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in Chlamydomonas reinhardtii using the phosphorylation status of its putative downstream target, CrS6K. Using this assay, we probe the modulation of cellular TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy, and nitrogen (N) starvation. Importantly, we uncover that excess acetate in the medium leads to high cellular reactive oxygen species levels, triggering autophagy and a concomitant drop in TOR kinase activity in a dose-dependent manner, thus leading to a N-starvation-like cellular phenotype, even when nitrogen is present.

Physiological and Ecological Aspects of Chlorella sorokiniana (Trebouxiophyceae) Under Photoautotrophic and Mixotrophic Conditions.

Mixotrophy is a metabolic strategy in which an organism is autotrophic and heterotrophic simultaneously. Considering that the aquatic environment provides several organic sources of carbon, it is probably common for microalgae to perform mixotrophy and not only photoautotrophy, but little is known about microalgae mixotrophy. The present work aimed at investigating the growth, photosynthetic activity, morphology, and biochemical composition of the microalga Chlorella sorokiniana in mixotrophic and photo-mixotrophic conditions, comparing it with photoautotrophy. The results showed pH changes after glucose addition, reaching pH 11.62 in mixotrophic and 10.47 in sequential photo-mixotrophic cultures, which limited the microalgal growth. Highest biomass was obtained in the mixotrophic culture in comparison with the sequential photo-mixotrophic one. Rapid light saturation curves showed that α (photosynthetic efficiency, 1.69) and relative electron transport rate (rETR; 565.61) were higher in the mixotrophic cultures, whereas the highest Ik (irradiance saturation, 386.68) was obtained in the photoautotrophic ones. In the sequential photo-mixotrophic cultures, photosynthetic activity varied during glucose consumption, decreasing the maximum quantum yield Fv/Fm after glucose addition, indicating change in metabolism, from photoautotrophy to mixotrophy by the microalga. The results showed that the mixotrophic cultures had higher production of chlorophyll a (6.26 mg mL-1), cell density (6.62 × 107 cell mL-1), and lipids (0.06 pg µm-3). Sequential photo-mixotrophic cultures showed the highest biovolume (360.5 µm3 cell-1) and total carbohydrates (0.026 pg µm-3). The protein concentration was 3.2 and 2.4 times higher in photoautotrophy and photo-mixotrophic growth, respectively, than in mixotrophy, but lipids were three times higher under mixotrophy. The biochemical changes we observed indicate that the microalga's plasticity in face of new environmental characteristics, such as the presence of organic carbon, can change the flow of energy through natural ecosystems.

[Diversity and Physiological and Biochemical Properties of Heterotrophic Bacteria. Isolated from Lake Baikal Neuston.]

For heterotrophic microorganisms (44 strains) isolated-from the surface film of Lake Baikal, iden- tification was carried out and their. physiological and biochemical characteristics were determined. Com- pared to the water column, diversity of cultured heterotrophs was low, indicating formation of stable micro- bial communities at the air-water interphase interface. Heterotrophic bacteria isolated from the surface mi- crolayer exhibited the enzymatic activity comparable to that for strains form other biofilm associations. Deinococcusfi6us strain NA202 'vas the most active component of the community, capable of utilization of the broadest spectrum of mono- and disaccharides,'sugars, and amino acids. This strain possessed the highest diversity of extracellular enzymes and was the most resistant to UV radiation. The physiological and bio- chemical properties of this strain may-be responsible for its adaptation to survival in extreme conditions of the surface microlayer. Our results improve our understanding of occurrence of UV-resistant strains in freshwater ecosystems.

An efficient screening method for the isolation of heterotrophic bacteria influencing growth of diatoms under photoautotrophic conditions.

Interactions between photoautotrophic diatoms and heterotrophic bacteria are important for the biogeochemical C-cycle in the oceans. Additionally, biofilms formed by diatoms and bacteria are the initiating step of biofouling processes, which causes high costs in shipping. Despite this ecological and economical importance, the knowledge about biochemical and molecular mechanisms underlying these interkingdom interactions is relatively small. For analyzing these mechanisms, laboratory model systems are required. In this study, an efficient screening method for isolating bacteria influencing photoautotrophic diatom growth was established. First, diatom cultures of Phaeodactylum tricornutum and Thalassiosira pseudonana were made axenic by applying ß-lactam antibiotics. Second, a non-invasive method for measuring growth of multiple parallel diatom cultures by chlorophyll fluorescence was established. This method allowed semi-quantitative chlorophyll determination of cultures with up to 3 µg (chlorophyll) ml(-1). Axenic diatom cultures were then used for enriching bacteria and led to the isolation of 24 strains influencing growth of both diatom strains in various ways. For example, Rheinheimera sp. strain Tn16 inhibited growth of T. pseudonana, while it stimulated growth and cell aggregation of P. tricornutum. Thus, this screening method is appropriate for isolating heterotrophic bacteria showing different interactions with different diatom species ranging from synergistic to antagonistic. In consecutive applications, this method will be useful to screen for bacterial mutants with altered phenotypes regarding the influence on diatom growth.

Subunit Q Is Required to Stabilize the Large Complex of NADPH Dehydrogenase in Synechocystis sp. Strain PCC 6803.

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1, NdhF1, and NdhP, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, NDH-1-dependent cyclic electron transport around photosystem I, and CO2 uptake. Two mutants sensitive to high light for growth and impaired in cyclic electron transport around photosystem I were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in an open reading frame encoding a product highly homologous to NdhQ, a single-transmembrane small subunit of the NDH-1L complex, identified in Thermosynechococcus elongatus by proteomics strategy. Deletion of ndhQ disassembled about one-half of the NDH-1L to NDH-1M and consequently impaired respiration, but not CO2 uptake. During prolonged incubation of the thylakoid membrane with n-dodecyl-ß-D-maltoside at room temperature, the rest of the NDH-1L in ΔndhQ was disassembled completely to NDH-1M and was much faster than in the wild type. In the ndhP-deletion mutant (ΔndhP) background, absence of NdhQ almost completely disassembled the NDH-1L to NDH-1M, similar to the results observed in the ΔndhD1/ΔndhD2 mutant. We therefore conclude that both NdhQ and NdhP are essential to stabilize the NDH-1L complex.

Prospective technology on bioethanol production from photofermentation.

The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by microalgae represent a process with reduced environmental impact with efficient CO2 fixation and requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas reinhardtii through the cellular metabolism in a light/dark cycle at 25 °C in a TAP medium with sulfur depletion. The parameters evaluated were inoculum concentration and the medium supplementation with mixotrophic carbon sources. The combination of C.reinhardtii and Rhodobacter capsulatus through a hybrid or co-culture systems was also investigated as well. C.reinhardtii maintained in TAP-S produced 19.25±4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with milk whey permeated and the algal effluent used by R. capsulatus, the ethanol production achieved 19.94±2.67 g/L.

Regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime.

Proteomics in conjunction with biochemical strategy was employed to unravel regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime (HPC). Interestingly, HPC triggered transiently synthesis of starch followed by substantial lipid accumulation. And a marked decrease in intracellular protein and chlorophyll contents was also observed after 12h of photo-induction. The highest lipid content of 50.5% was achieved upon the photo-induction stage, which represented 69.3% higher than that of the end of heterotrophic cultivation. Results suggested that turnover of carbon-nitrogen-rich compounds such as starch, protein, and chlorophyll might provide carbon or energy for lipid accumulation. The proteomics analysis indicated that several pathways including glycolysis, TCA cycle, ß-oxidation of fatty acids, Calvin cycle, photosynthesis, energy and transport, protein biosynthesis, regulate and defense were involved in the lipid biosynthesis. Malate dehydrogenase and acyl-CoA dehydrogenase were suggested as key regulatory factors in enhancing lipid accumulation.

Photosynthetic light reactions increase total lipid accumulation in carbon-supplemented batch cultures of Chlorella vulgaris.

Microalgae are an attractive biofuel feedstock because of their high lipid to biomass ratios, lipid compositions that are suitable for biodiesel production, and the ability to grow on varied carbon sources. While algae can grow autotrophically, supplying an exogenous carbon source can increase growth rates and allow heterotrophic growth in the absence of light. Time course analyses of dextrose-supplemented Chlorella vulgaris batch cultures demonstrate that light availability directly influences growth rate, chlorophyll production, and total lipid accumulation. Parallel photomixotrophic and heterotrophic cultures grown to stationary phase reached the same amount of biomass, but total lipid content was higher for algae grown in the presence of light (an average of 1.90 mg/mL vs. 0.77 mg/mL over 5 days of stationary phase growth).

Cyanobacterial phytochrome2 regulates the heterotrophic metabolism and has a function in the heat and high-light stress response.

Cyanobacteria combine the photosynthetic and respiratory electron transport in one membrane system, the thylakoid membrane. This feature requires an elaborate regulation mechanism to maintain a certain redox status of the electron transport chain, hence allowing proper photosynthetic and respiratory energy metabolism. In this context, metabolic adaptations, as seen in the light-to-dark and dark-to-light transitions, are particularly challenging. However, the molecular basis of the underlying regulatory mechanisms is not well-understood. Here, we describe a function of cyanobacterial phytochrome2 (Cph2), a phytochrome of the cyanobacterial model system Synechocystis sp. PCC 6803, in regulation of the primary energy metabolism. When cells are shifted from photoautotrophic planktonic growth to light-activated heterotrophic growth and biofilm initiation, knockout of Cph2 results in impaired growth, a decrease in the activity of Glc-6-P dehydrogenase, a decrease of the transcript abundance/activity of cytochrome-c-oxidase, and slower phycocyanin degradation. Measurements of the plastoquinone reduction confirm an impaired heterotrophic metabolism in the cph2 knockout. When cells that were adapted to heterotrophic metabolism are shifted back to light conditions, the knockout of Cph2 results in an altered photosystem II chlorophyll fluorescence induction curve, which is indicative of an impaired redox balance of the electron transport chain. Moreover, Cph2 plays a role in the heat and high-light stress response, particularly under photomixotrophic conditions. Our results show a function of Cph2 in the adaptation of the primary energy metabolism to changing trophic conditions. The physiological role of Cph2 in biofilm formation is discussed.

Shifts in microbial community structure and function in light- and dark-grown biofilms driven by warming.

Biofilms are dynamic players in biogeochemical cycling in running waters and are subjected to environmental stressors like those provoked by climate change. We investigated whether a 2°C increase in flowing water would affect prokaryotic community composition and heterotrophic metabolic activities of biofilms grown under light or dark conditions. Neither light nor temperature treatments were relevant for selecting a specific bacterial community at initial phases (7-day-old biofilms), but both variables affected the composition and function of mature biofilms (28-day-old). In dark-grown biofilms, changes in the prokaryotic community composition due to warming were mainly related to rotifer grazing, but no significant changes were observed in functional fingerprints. In light-grown biofilms, warming also affected protozoan densities, but its effect on prokaryotic density and composition was less evident. In contrast, heterotrophic metabolic activities in light-grown biofilms under warming showed a decrease in the functional diversity towards a specialized use of several carbohydrates. Results suggest that prokaryotes are functionally redundant in dark biofilms but functionally plastic in light biofilms. The more complex and self-serving light-grown biofilm determines a more buffered response to temperature than dark-grown biofilms. Despite the moderate increase in temperature of only 2°C, warming conditions drive significant changes in freshwater biofilms, which responded by finely tuning a complex network of interactions among microbial populations within the biofilm matrix.

Thylakoid membrane maturation and PSII activation are linked in greening Synechocystis sp. PCC 6803 cells.

Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, "greening" of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.

Environmental pH affects photoautotrophic growth of Synechocystis sp. PCC 6803 strains carrying mutations in the lumenal proteins of PSII.

Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5.

Enhancement of microalgal biomass and lipid productivities by a model of photoautotrophic culture with heterotrophic cells as seed.

For overcoming the long period of seed cultured photoautotrophically and inadequate cell supply for the inoculation of microalgae photoautotrophic cultivation, a model for the photoautotrophic culture of three Chlorella species with heterotrophic cells as seed was investigated. The model can not only take advantages of rapid cell growth in heterotrophic process for preparation of cells as seed but also increase the biomass and lipid productivities of the microalgae cultivated photoautotrophically. The results showed that biomass productivities of Chlorella pyrenoidosa, Chlorella ellipsoidea and Chlorella vulgaris cultured by heterotrophy were 20.9, 26.9 and 25.2 times higher than those by photoautotrophy in seed culturing period. In the subsequent photoautotrophic culture, the biomass and lipid productivities of C. pyrenoidosa, C. ellipsoidea and C. vulgaris with heterotrophic seed were 1.91, 1.51, 1.48 and 1.66, 1.37, 1.42 times higher than those with photoautotrophic seed. Furthermore, the culture model was also carried out successfully outdoor.

Growth of Dunaliella tertiolecta and associated bacteria in photobioreactors.

The aim of this study was to test three flat-plate photobioreactor configurations for cultivation of marine green alga Dunaliella tertiolecta under non-axenic growth conditions and to characterize and quantify the associated bacteria. The photobioreactor cultivations were conducted using tap water-based media. Static mixers intended to enhance mixing and light utilization did not generally increase algal growth at the low light intensities used. The maximum biomass concentration (measured as volatile suspended solids) and maximum specific growth rate achieved in the flat plate with no mixer were 2.9 g l⁻¹ and 1.3 day⁻¹, respectively. Based on quantitative polymerase chain reaction, bacterial growth followed the growth of D. tertiolecta. Based on 16S rDNA amplification and denaturing gradient gel electrophoresis profiling, heterotrophic bacteria in the D. tertiolecta cultures mainly originated from the non-axenic algal inocula, and tap water heterotrophs were not enriched in high chloride media (3 % salinity). Bacterial communities were relatively stable and reproducible in all flat-plate cultivations and were dominated by Gammaproteobacteria, Flavobacteria, and Alphaproteobacteria.

Comparison of Chloroflexus aurantiacus strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically.

Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography-mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under photoheterotrophic culture condition. Fifty-four of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO(2) fixation pathway, and components of electron transport chains. The remaining 188 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.

The role of heterotrophic carbon acquisition by the hemiparasitic plant Rhinanthus alectorolophus in seedling establishment in natural communities: a physiological perspective.

• Heterotrophic acquisition of substantial amounts of organic carbon by hemiparasitic plants was clearly demonstrated by numerous studies. Many hemiparasites are, however, also limited by competition for light preventing the establishment of their populations on highly productive sites. • In a growth-chamber experiment, we investigated the effects of competition for light, simulated by shading, on growth and heterotrophic carbon acquisition by the hemiparasite Rhinanthus alectorolophus attached to C(3) and C(4) hosts using analyses of biomass production and stable isotopes of carbon. • Shading had a detrimental effect on biomass production and vertical growth of the hemiparasites shaded from when they were seedlings, while shading imposed later caused only a moderate decrease of biomass production and had no effect on the height. Moreover, shading increased the proportion of host-derived carbon in hemiparasite biomass (up to 50% in shaded seedlings). • These results demonstrate that host-derived carbon can play a crucial role in carbon budget of hemiparasites, especially if they grow in a productive environment with intense competition for light. The heterotrophic carbon acquisition can allow hemiparasite establishment in communities of moderate productivity, helping well-attached hemiparasites to escape from the critical seedling stage.

Interspecies variation in survival and growth of filamentous heterotrophic bacteria in response to UVC radiation.

Ultraviolet radiation is an important environmental constraint on the evolution of life. In addition to its harmful effects, ultraviolet radiation plays an important role in generating genetic polymorphisms and acting as a selective agent. Understanding how prokaryotes cope with high radiation can give insights on the evolution of life on Earth. Four representative filamentous bacteria from the family Cytophagaceae with different pigmentation were selected and exposed to different doses of UVC radiation (15-32,400Jm(-2)). The effect of UVC radiation on bacterial survival, growth and morphology were investigated. Results showed high survival in response to UVC for Rudanella lutea and Fibrisoma limi, whereas low survival was observed for Fibrella aestuarina and Spirosoma linguale. S. linguale showed slow growth recovery after ultraviolet exposure, R. lutea and F. limi showed intermediate growth recovery, while F. aestuarina had the fastest recovery among the four tested bacteria. In terms of survival, S. linguale was the most sensitive bacterium whereas R. lutea and F. limi were better at coping with UVC stress. The latter two resumed growth even after 2h exposure (∼10,800Jm(-2)). Additionally, the ability to form multicellular filaments after exposure was tested using two bacteria: one representative of the high (R. lutea) and one of the low (F. aestuarina) survival rates. The ability to elongate filaments due to cell division was preserved but modified. In R. lutea 10min exposure reduced the average filament length. The opposite was observed in F. aestuarina, where the 5 and 10min exposures increased the average filament length. R. lutea and F. limi are potential candidates for further research into survival and resistance to ultraviolet radiation stress.

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions.

We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 microM DCMU. This mutant carries an R7L mutation on the alpha-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 A PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lalpha cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lalpha cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lalpha cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the alpha-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.

Self-sustained phototrophic microbial fuel cells based on the synergistic cooperation between photosynthetic microorganisms and heterotrophic bacteria.

A sediment-type self-sustained phototrophic microbial fuel cell (MFC) was developed to generate electricity through the synergistic interaction between photosynthetic microorganisms and heterotrophic bacteria. Under illumination, the MFC continuously produced electricity without the external input of exogenous organics or nutrients. The current increased in the dark and decreased with the light on, possibly because of the negative effect of the oxygen produced via photosynthesis. Continuous illumination inhibited the current production while the continuous dark period stimulated the current production. Extended darkness resulted in a decrease of current, probably because of the consumption of the organics accumulated during the light phase. Using color filters or increasing the thickness of the sediment resulted in a reduction of the oxygen-induced inhibition. Molecular taxonomic analysis revealed that photosynthetic microorganisms including cyanobacteria and microalgae predominated in the water phase, adjacent to the cathode and on the surface of the sediment. In contrast, the sediments were dominated by heterotrophic bacteria, becoming less diverse with increasing depth. In addition, results from the air-cathode phototrophic MFC confirmed the light-induced current production while the test with the two-chamber MFC (in the dark) indicated the presence of electricigenic bacteria in the sediment.