Photoautotrophic Growth Research Articles

De-etiolation-induced protein 1 (DEIP1) mediates assembly of the cytochrome b6f complex in Arabidopsis

The conversion of light energy to chemical energy by photosynthesis requires the concerted action of large protein complexes in the thylakoid membrane. Recent work has provided fundamental insights into the three-dimensional structure of these complexes, but how they are assembled from hundreds of parts remains poorly understood. Particularly little is known about the biogenesis of the cytochrome b6f complex (Cytb6f), the redox-coupling complex that interconnects the two photosystems. Here we report the identification of a factor that guides the assembly of Cytb6f in thylakoids of chloroplasts. The protein, DE-ETIOLATION-INDUCED PROTEIN 1 (DEIP1), resides in the thylakoid membrane and is essential for photoautotrophic growth. Knock-out mutants show a specific loss of Cytb6f, and are defective in complex assembly. We demonstrate that DEIP1 interacts with the two cytochrome subunits of the complex, PetA and PetB, and mediates the assembly of intermediates in Cytb6f biogenesis. The identification of DEIP1 provides an entry point into the study of the assembly pathway of a crucial complex in photosynthetic electron transfer.

Evaluation of the phototrophic growth of Haematococcus pluvialis under outdoor lighting conditions inside a bubble column reactor at a laboratory scale

Haematococcus pluvialis is a green microalga of commercial and industrial interest due to its ability to accumulate high amounts of astaxanthin. In the present work, the photoautotrophic growth of a local strain of H. pluvialis is evaluated and analysed under illuminating conditions corresponding to a bubble column reactor located in Santa Fe, Argentina. Lighting conditions were reproduced using a previously developed methodology published by our research group, based on solid and rigorous radiation transfer modelling between surfaces. The latter implies using a commercial reactor with a custom-designed LED module attached to deliver the same amount of PAR radiant energy per time and volume unit (volumetric radiant flux, [μmol s−1 L−1]). To this end, typical winter, summer and autumn days were chosen because they differ in the photoperiod, the amount of daily light reaching the reactor, and the hourly values of volumetric radiant flow. In the cultivation experiments, the reactor was operated in batch mode, inoculated with aplanospore cells, and the composition and pH of the medium were allowed to evolve freely.Our data is in agreement with the notion that the lighting profile significantly affects biomass productivity and total carotenoids content. Consistently, life cycle duration and the amount of biomass and pigments produced have changed as well.

Enhanced photoautotrophic growth of Chlorella vulgaris in starch wastewater through photo-regulation strategy

Microalgae biomass production with starch wastewater (SW) is a promising approach to realize waste recovery and cost reduction due to the inherent copious nutrients and nontoxic compounds in SW. However, the application of this technique is significantly hindered by low biomass production on account of the poor photosynthetic efficiency of microalgae. In this regard, we proposed a photo-regulation strategy characterized by the adjusting of numbers of light/dark (L/D) cycles, and compositions of light wavelength, which was proved to be an effective method for stimulating intracellular photo electron transfer and enhancing photosynthetic efficiency, to boost microalgae biomass accumulation. Additionally, responses of the microalgae photo-biochemical conversion, and the wastewater treatment performance at various number of L/D cycles and light wavelengths were discussed. The experimental results indicated that the biomass production increased when the L/D period was increased from 2 h:2 h–12 h:12 h. When the L/D period was 2 h:2 h, the biomass production reached a maximum value of 1.28 g L−1, which was 19.6% higher than that of the control group when the L/D period was 12 h:12 h. Furthermore, with respect to microalgae growth under monochromatic light, the maximum biomass concentration (1.25 g L−1) and lipid content (32.2%) of Chlorella were achieved under blue light; whereas, the minimum values were attained under red light (1.05 g L−1 and 19.3%, respectively). When the red light and blue light were mixed and supplied, the microalgae biomass productivity was higher than that under white light, and the highest lipid productivity was 109.0 mg−1 L−1 d under a blue: red ratio of 2:1. Moreover, gas chromatography analysis demonstrated that the methyl in the range of C16–C18 in the system was higher than 70%. Fatty acid methyl esters (FAMEs) containing palmitic acid (C16:0) and oleic acid (C18:1) are beneficial for production of biodiesel, and the quality of fatty acid methyl ester used in biodiesel production can be improved using microalgae cultured under the mixed wavelengths of blue and red. Finally, Chlorella was cultured in PBR and reached the peak concentration of 2.45 g L−1 by semi-continuous process with the HRT regulation.

Possibilities of using thermal and mineral springs of the Republic of Kazakhstan as a medium for the cultivation of microalgae – biodiesel producers

Microalgae are characterized by rapid photoautotrophic growth and a high rate of biomass accumulation and are considered as an alternative feedstock for renewable energy sources such as biodiesel. Biofuel research is not just a matter of finding the right type of biomass and converting it into fuel, but also finding environmentally and economically viable options. The paper presents the results of studies on the use of thermal and mineral water sources as a medium for the cultivation of microalgae Parachlorella kessleri DZP-5, Parachlorella kessleri B2 and Ankistrodesmus falcatus B5. It was shown that when using water from the «Arshan» thermal water source and the Balkhash salt lake to dilute the Tamiya medium in a ratio of 50:50, it is possible to obtain an intensive growth of microalgae cultures and at the same time preserve the main characteristics of microalgae producers of biodiesel fuel. The composition of fatty acids of three tested strains of microalgae is also given, which showed their potential as producers of biodiesel. Key words: microalgae, thermal and mineral springs, cultivation, biodiesel producers, fatty acids.

Characteristics of Chemical Solutes and Mineral Dust in Ice of the Ablation Area of a Glacier in Tien Shan Mountains, Central Asia

Diverse microbial communities live on glacial surfaces, with abundances and diversities dictated by the chemical and physical conditions of the supraglacial environment. Chemical solutes and mineral dust on glacial surfaces are generally derived from the atmosphere as aerosols, but they can also be supplied from glacial ice. In this study, a 56-m ice core from the ablation area of a mountain glacier in the Tien Shan Mountains of Central Asia was analyzed to characterize the chemical solutes and mineral dust in glacial ice. Soluble chemical ion and mineral dust analysis in the ice core showed that their concentrations varied with depth. The most dominant ion was Ca2+ (mean: 79.8 μEq L−1), followed by Cl−, SO42-, NH4+, and NO3−. The mean dust concentration in the glacial ice was 2.5 ×105 number ml−1. Chemical solute and mineral dust concentrations in the samples indicate that melting glacial ice could potentially affect supraglacial conditions. The annual fluxes of the major ions outcropping from glacial ice were estimated based on their concentrations and the observed melt rate of the ice surface. Consequently, they were comparable to or higher than those from the atmosphere. The mean mass flux of mineral dust from glacial ice was greater than that from the atmosphere. Our results showed that glacial ice supplies chemical solutes and mineral dust to the supraglacial environment and that changing melting rates of glacial ice would affect the chemical conditions on the glacier surface and the growth of photoautotrophs on the ablating ice surface.

Combinatorial Engineering Enables Photoautotrophic Growth in High Cell Density Phosphite-Buffered Media to Support Engineered Chlamydomonas reinhardtii Bio-Production Concepts.

Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome modification and transgene expression are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we used combinatorial chloroplast and nuclear genome engineering to augment the metabolism of the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as sole sources of phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combined previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products from green microalgae.

Effects of mutations of D1-R323, D1-N322, D1-D319, D1-H304 on the functioning of photosystem II in Thermosynechococcus vulcanus.

Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63-91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ.

TCP15 interacts with GOLDEN2-LIKE 1 to control cotyledon opening in Arabidopsis.

After germination, exposure to light promotes the opening and expansion of the cotyledons and the development of the photosynthetic apparatus in a process called de-etiolation. This process is crucial for seedling establishment and photoautotrophic growth. TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are important developmental regulators of plant responses to internal and external signals that are grouped into two main classes. In this study, we identified GOLDEN2-LIKE 1 (GLK1), a key transcriptional regulator of photomorphogenesis, as a protein partner of class I TCPs during light-induced cotyledon opening and expansion in Arabidopsis. The class I TCP TCP15 and GLK1 are mutually required for cotyledon opening and the induction of SAUR and EXPANSIN genes, involved in cell expansion. TCP15 also participates in the expression of photosynthesis-associated genes regulated by GLK1, like LHCB1.4 and LHCB2.2. Furthermore, GLK1 and TCP15 bind to the same promoter regions of different target genes containing either GLK or TCP binding motifs and binding of TCP15 is affected in a GLK1-deficient background, suggesting that a complex between TCP15 and GLK1 participates in the induction of these genes. We postulate that GLK1 helps to recruit TCP15 for the modulation of cell expansion genes in cotyledons and that the functional interaction between these transcription factors may serve to coordinate the expression of cell expansion genes with that of genes involved in the development of the photosynthetic apparatus.

Elevated temperature mitigates the prolonged effect of high nitrogen on Microcystis aeruginosa removal through mixotrophic Ochromonas gloeopara grazing

Cyanobacterial blooms are increasingly threatening the aquatic ecosystem functioning as a result of the global warming and eutrophication. The “top-down” control of cyanobacteria from consumers like the protozoans shows great potential because of the effectiveness and environment-friendliness. To reveal how the nutrition availability and elevated temperature affect the cyanobacteria removal through protozoans grazing, we grew the toxic Microcystis aeruginosa and the mixotrophic Ochromonas gloeopara in monocultures and cocultures at environmentally relevant nitrogen levels (0.5–8.0 mg L−1) under 25 °C and 30 °C, respectively. The growth of M. aeruginosa in monocultures was significantly enhanced as nitrogen concentration and temperature rose, partially benefitting from the promoted photosynthesis. By contrast, nitrogen availability affected neither the photoautotrophic growth nor the feeding on Microcystis of the mixotrophic O. gloeopara, but high temperature induced the mixotroph to be more heterotrophic as evidenced by the suppressed photosynthesis but strengthened feeding activity. Accordingly, the M. aeruginosa removal through O. gloeopara grazing in cocultures was delayed with increasing nitrogen, which, however, was sharply accelerated by elevated temperature. Based on the Gaussian models fitting, the theoretical time that the Microcystis was removed at 25 °C was prolonged from about 7.5 days to 10 days with increased nitrogen, but it was reduced to less than 4.6 days in all groups at 30 °C. While the intensity of Microcystis blooms is strongly positively correlated to the nutrition availability and temperature, the present study provided references for the practical application of Microcystis removal through grazing outdoors.

Elioraea tepida, sp. nov., a Moderately Thermophilic Aerobic Anoxygenic Phototrophic Bacterium Isolated from the Mat Community of an Alkaline Siliceous Hot Spring in Yellowstone National Park, WY, USA.

Strain MS-P2T was isolated from microbial mats associated with Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, WY, USA. The isolate grows chemoheterotrophically by oxygen-dependent respiration, and light stimulates photoheterotrophic growth under strictly oxic conditions. Strain MS-P2T synthesizes bacteriochlorophyll a and the carotenoid spirilloxanthin. However, photoautotrophic growth did not occur under oxic or anoxic conditions, suggesting that this strain should be classified as an aerobic anoxygenic phototrophic bacterium. Strain MS-P2T cells are motile, curved rods about 0.5 to 1.0 μm wide and 1.0 to 1.5 μm long. The optimum growth temperature is 45–50 °C, and the optimum pH for growth is circum-neutral (pH 7.0–7.5). Sequence analysis of the 16S rRNA gene revealed that strain MS-P2T is closely related to Elioraea species, members of the class Alphaproteobacteria, with a sequence identity of 96.58 to 98%. The genome of strain MS-P2T is a single circular DNA molecule of 3,367,643 bp with a mol% guanine-plus-cytosine content of 70.6%. Based on phylogenetic, physiological, biochemical, and genomic characteristics, we propose this bacteriochlorophyll a-containing isolate is a new species belonging to the genus Elioraea, with the suggested name Elioraea tepida. The type-strain is strain MS-P2T (= JCM33060T = ATCC TSD-174T).

Determination of photoautotrophic growth and inhibition kinetics by the Monod and the Aiba models and bioenergetics of local microalgae strain

The usage of fossil fuels results in a high amount of greenhouse gas (GHG) emissions and renewable green energy requirements entail saving ecological balance. Therefore, microalgae cultivation is widespread as a suitable raw material to produce renewable and sustainable fuel. Mathematical models are useful tools for the estimation of different conditions of a system. In this study, mathematical models were developed for monitoring the cultivation of local species of microalgae based on the chlorophyll-a and biomass concentration. Coefficients that were calculated from the Monod kinetic model were μmax; 0.03 day−1, KS, Ci; 0.53 mM with an R2 value of 0.93 and from the Aiba inhibition kinetic model was μmax and KS, Ci 1.48 day−1 and 0.08 mM with an R2 value of 0,73. According to the literature, there was no model was developed for the determination of kinetic coefficients based on chlorophyll-a production due to the inorganic carbon consumption. While both growth and inhibition models were developed for the inorganic carbon consumption, chlorophyll-a concentration was used for the growth model and biomass concentration was used for the inhibition model which caused and directly affected by the decrease of light penetration. The maximum biomass and chlorophyll-a concentrations were found as 1.2 g/L and 27.8 mg/L respectively with 10.24 mg/L. day−1 nitrogen and 1.19 mg/L.day−1 phosphorus uptake rate.

Photosynthetic Conversion of CO2 Into Pinene Using Engineered Synechococcus sp. PCC 7002

Metabolic engineering of cyanobacteria has received much attention as a sustainable strategy to convert CO2 to various longer carbon chain fuels. Pinene has become increasingly attractive since pinene dimers contain high volumetric energy and have been proposed to act as potential aircraft fuels. However, cyanobacteria cannot directly convert geranyl pyrophosphate into pinene due to the lack of endogenous pinene synthase. Herein, we integrated the gene encoding Abies grandis pinene synthase into the model cyanobacterium Synechococcus sp. PCC 7002 through homologous recombination. The genetically modified cyanobacteria achieved a pinene titer of 1.525 ± 0.l45 mg L−1 in the lab-scale tube photobioreactor with CO2 aeration. Specifically, the results showed a mixture of α- and β-pinene (∼33:67 ratio). The ratio of β-pinene in the product was significantly increased compared with that previously reported in the engineered Escherichia coli. Furthermore, we investigated the photoautotrophic growth performances of Synechococcus overlaid with different concentrations of dodecane. The work demonstrates that the engineered Synechococcus is a suitable potential platform for β-pinene production.

Flux balance analysis for overproduction of organic acids by Synechocystis sp. PCC 6803 under dark anoxic condition

Cyanobacteria have recently been considered as potential organisms for metabolic engineering with an objective to improve its ability to synthesize biofuel precursors and other value-added products. A modified version of the genome-scale metabolic model of Synechocystis sp. PCC 6803 model organism was used to theoretically demonstrate the flux distribution in the central carbon metabolism towards optimal accumulation of organic acids, namely succinate, acetate, lactate and malate. We further validate the model using experimental data reported by Hasunuma et al. Met. Engg. Comm., 2016, 3, 130–141 for organic acid synthesis under dark anoxic conditions from glycogen accumulated during photoautotrophic growth conditions by sequestering atmospheric CO2. We show that key limiting factors are redox balance causing gluconeogenesis, availability of CO2 and channeling of carbon flux to TCA cycle, in addition to the rate of breakdown of glycogen. Sodium bicarbonate supplementation enhances succinate production flux by eliminating the CO2 limitations for the phosphoenolpyruvate (PEP) to oxaloacetate biochemical reaction catalyzed by PEP carboxylase. A combined strategy of supplementation with NaHCO3 and increased temperature can only offer ~25% of the theoretical maximum succinate production. We suggest possible metabolic pathway interventional approaches to improve the succinate productivity.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Tandem gene amplification restores photosystem II accumulation in cytochrome b559 mutants of cyanobacteria.

Summary Cytochrome (Cyt) b 559 is a key component of the photosystem II complex (PSII) that is essential for its proper functioning and assembly. Site‐directed mutants of the model cyanobacterium Synechocystis sp. PCC6803 with mutated heme axial ligands of Cyt b 559 have little PSII and are therefore unable to grow photoautotrophically.Here we describe two types of Synechocystis autotrophic transformants that retained the same mutations in Cyt b 559 but are able to accumulate PSII and grow photoautotrophically. Whole‐genome sequencing revealed that all of these autotrophic transformants carried a variable number of tandem repeats (from 5 to 15) of chromosomal segments containing the psbEFLJ operon.RNA‐seq analysis showed greatly increased transcript levels of the psbEFLJ operon in these autotrophic transformants. Multiple copies of the psbEFLJ operon in these transformants were only maintained during autotrophic growth, while its copy numbers gradually decreased under photoheterotrophic conditions. Two‐dimensional PAGE analysis of membrane proteins revealed a strong deficiency in PSII complexes in the Cyt b 559 mutants that was reversed in the autotrophic transformants.These results illustrate how tandem gene amplification restores PSII accumulation and photoautotrophic growth in Cyt b 559 mutants of cyanobacteria, and may serve as an important adaptive mechanism for cyanobacterial survival.

Effects of ammonium concentration in the catholyte on electricity and algal biomass generation in a photosynthetic microbial fuel cell treating wastewater

In this study, algal cathodic compartments were integrated to generate oxygen in microbial fuel cells. Chlorella vulgaris and Scenedesmus quadricauda were grown under three NH4+–N concentrations (50, 300, and 900 mg L−1) in the catholyte. With 50 mg L−1 of NH4+–N, the highest maximum power densities of 16.72 and 5.96 mW m−2 were obtained with Chlorella vulgaris and Scenedesmus quadricauda, respectively. Under this condition, dissolved oxygen reached 19.27 and 9.87 mg L−1 for Chlorella vulgaris and Scenedesmus quadricauda respectively, along with 100% removal of NH4+–N within 210 h. Both cultures showed inhibition under higher concentrations of NH4+–N, resulting in decreased photoautotrophic growth that generates oxygen for the cathodic reaction. Under all concentrations, Chlorella vulgaris displayed better tolerance to ammonium. From the results, it can be summarized that 50 mg L−1 of NH4+–N in wastewater is suitable as a microalgal cultivation medium in a cathodic compartment.

Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria.

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

CO2 enrichment alters morphophysiology and improves growth and acclimatization in Etlingera Elatior (Jack) R.M. Smith micropropagated plants

Conventional in vitro propagation may affect normal morphophysiological characteristics and survival during the acclimatization of commercial plants, such as Etlingera Elatior (Jack) R.M. Smith. CO2 enrichment, gas-permeable membranes, and reduced carbohydrate concentration in culture media can be used to mitigate these effects. Here, we aimed to determine the impact of photomixotrophic and photoautotrophic growth on morphophysiological responses of E. elatior and its survival during ex vitro acclimatization. In vitro-established plants were placed in flasks containing semisolid MS medium with 0.54 µM α-naphthalene acetic acid and sucrose (0 or 15 g L−1). The flasks were sealed with lids with or without orifices covered by gas-permeable membranes and kept for 45 days under forced-air ventilation (360 or 1000 µmol−1 CO2). Growth variables, anatomical and physiological characteristics, and survival rate were evaluated. Overall, 1000 µmol mol−1 CO2, gas-permeable membranes, and 15 g L−1 sucrose resulted in plants with higher fresh and dry mass, higher level of photosynthetic pigments, as well as more developed secondary ribs in the leaves, prominent midribs, highly organized and differentiated chlorenchyma cells (palisade and spongy parenchyma), more developed vascular bundles, and intercellular gaps were reduced, compared to conventionally in vitro-propagated plants. Moreover, these conditions promote morphophysiological responses and favored survival of E. elatior plants (75%) when transferred to ex vitro conditions.

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii.

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.

Techno-economic and life-cycle assessment of fuel production from mixotrophic Galdieria sulphuraria microalgae on hydrolysate

Outdoor photoautotrophic algal growth is limited by light attenuation and attendant respiratory CO2 losses during dark periods, limiting its productivity potential and carbon use efficiency. Developing a system that leverages mixotrophic growth (combining the benefits of both heterotrophic and photoautotrophic growth) has the potential to dramatically improve the total productivity and economics of the system. However, it is unknown if the productivity gains offset the added costs of outdoor mixotrophic cultivation using cellulosic hydrolysate as the feedstock. In this study, corn stover-derived cellulosic sugars were evaluated as the mixotrophic organic carbon source for the cultivation of Galdieria sulphuraria, which can metabolize both glucose and xylose from corn stover hydrolysate. A techno-economic analysis (TEA) and a life-cycle assessment (LCA) were conducted based on a detailed engineering process model for both glass helical photobioreactor and covered pond cultivation platforms, coupled with downstream conversion and upgrading to renewable diesel through hydrothermal liquefaction. Results show the minimum biomass selling price for cultivation in the photobioreactor design assuming a productivity of 1.575 kg m−3 day−1 and a substrate yield of 0.57 g g−1 is $2869 per dry metric ton. The costs are dramatically reduced in the covered pond design which assumes a productivity of 0.8 kg m−3 day−1 and a substrate yield of 0.7 g g−1, $921 per dry metric ton. Expanding the system boundary to include downstream processing results in a minimum fuel selling price of $8.24 and $3.32 dm3GE−1 for the photobioreactor and covered pond systems, respectively. Finally, life-cycle results demonstrate a global warming potential of 339 and 9.1 gCO2-eq. MJ−1 on a well-to-wheels basis and a net energy ratio of 2.21 and 0.25 MJ MJ−1 for the photobioreactor and covered pond systems, respectively. Discussion focuses on a required co-product selling price as a function of biomass diversion to meet economic parity with conventional fuels.