Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Structural and functional regulation of Chlamydomonas lysosome-related organelles during environmental changes.

Lysosome-related organelles (LROs) are a class of heterogeneous organelles conserved in eukaryotes that primarily play a role in storage and secretion. An important function of LROs is to mediate metal homeostasis. Chlamydomonas reinhardtii is a model organism for studying metal ion metabolism; however, structural and functional analyses of LROs in C. reinhardtii are insufficient. Here, we optimized a method for purifying these organelles from 2 populations of cells: stationary phase or overloaded with iron. The morphology, elemental content, and lysosomal activities differed between the 2 preparations, even though both have phosphorus and metal ion storage functions. LROs in stationary phase cells had multiple non-membrane-bound polyphosphate granules to store phosphorus. Those in iron-overloaded cells were similar to acidocalcisomes (ACs), which have a boundary membrane and contain 1 or 2 large polyphosphate granules to store more phosphorus. We established a method for quantifying the capacity of LROs to sequester individual trace metals. Based on a comparative proteomic analysis of these 2 types of LROs, we present a comprehensive AC proteome and identified 113 putative AC proteins. The methods and protein inventories provide a framework for studying the biogenesis and modification of LROs and the mechanisms by which they participate in regulating metal ion metabolism.

Biochemical and phylogenetic characterization of the wastewater tolerant Chlamydomonas biconvexa Embrapa|LBA40 strain cultivated in palm oil mill effluent.

The increasing demand for water, food and energy poses challenges for the world´s sustainability. Tropical palm oil is currently the major source of vegetable oil worldwide with a production that exceeds 55 million tons per year, while generating over 200 million tons of palm oil mill effluent (POME). It could potentially be used as a substrate for production of microalgal biomass though. In this study, the microalgal strain Chlamydomonas biconvexa Embrapa|LBA40, originally isolated from a sugarcane vinasse stabilization pond, was selected among 17 strains tested for growth in POME retrieved from anaerobic ponds of a palm oil industrial plant located within the Amazon rainforest region. During cultivation in POME, C. biconvexa Embrapa|LBA40 biomass productivity reached 190.60 mgDW • L-1 • d-1 using 15L airlift flat plate photobioreactors. Carbohydrates comprised the major fraction of algal biomass (31.96%), while the lipidic fraction reached up to 11.3% of dry mass. Reductions of 99% in ammonium and nitrite, as well as 98% reduction in phosphate present in POME were detected after 5 days of algal cultivation. This suggests that the aerobic pond stage, usually used in palm oil industrial plants to reduce POME inorganic load, could be substituted by high rate photobioreactors, significantly reducing the time and area requirements for wastewater treatment. In addition, the complete mitochondrial genome of C. biconvexa Embrapa|LBA40 strain was sequenced, revealing a compact mitogenome, with 15.98 kb in size, a total of 14 genes, of which 9 are protein coding genes. Phylogenetic analysis confirmed the strain taxonomic status within the Chlamydomonas genus, opening up opportunities for future genetic modification and molecular breeding programs in these species.

Outdoor pilot-scale raceway as a microalgae-bacteria sidestream treatment in a WWTP.

This study aims at demonstrating the feasibility of using microalgae-bacteria consortia for the treatment of the sidestream flow of the supernatant from blackwater dewatering (centrate) in an urban wastewater treatment plant in Northern Italy. A 1200 L raceway reactor was used for the outdoor cultivation of a diverse community of Chlorella spp., Scenedesmus spp. and Chlamydomonas spp. in continuous operation mode with 10 days hydraulic retention time. During the trial, an average daily areal productivity of 5.5 ± 7.4 g TSS m-2 day-1 was achieved while average nutrient removal efficiencies were 86% ± 7% and 71% ± 10% for NH4-N and PO4-P, respectively. The microalgal nitrogen assimilation accounted for 10% of the nitrogen in the centrate while 34% was oxidized to nitrite and nitrate. The oxygen produced by microalgae fully covert the oxygen demand for nitrification. This suggests that the proposed process would reduce the aeration demand for nitrification in the water line of the plant, while producing algal biomass to be further valorized for energy or material recovery.

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Truncated Hemoglobins 1 and 2 Are Implicated in the Modulation of Phosphorus Deficiency-Induced Nitric Oxide Levels in Chlamydomonas.

Truncated hemoglobins (trHbs) form a widely distributed family of proteins found in archaea, bacteria, and eukaryotes. Accumulating evidence suggests that trHbs may be implicated in functions other than oxygen delivery, but these roles are largely unknown. Characterization of the conditions that affect trHb expression and investigation of their regulatory mechanisms will provide a framework for elucidating the functions of these globins. Here, the transcription of Chlamydomonas trHb genes (THB1-12) under conditions of phosphorus (P) deprivation was analyzed. Three THB genes, THB1, THB2, and THB12 were expressed at the highest level. For the first time, we demonstrate the synthesis of nitric oxide (NO) under P-limiting conditions and the production of NO by cells via a nitrate reductase-independent pathway. To clarify the functions of THB1 and THB2, we generated and analyzed strains in which these THBs were strongly under-expressed by using an artificial microRNA approach. Similar to THB1 knockdown, the depletion of THB2 led to a decrease in cell size and chlorophyll levels. We provide evidence that the knockdown of THB1 or THB2 enhanced NO production under P deprivation. Overall, these results demonstrate that THB1 and THB2 are likely to contribute, at least in part, to acclimation responses in P-deprived Chlamydomonas.

Evidence of microalgal isotopic fractionation through enrichment of depleted uranium.

Resulting from the nuclear fuel cycle, large amounts of depleted uranium (DU) tails are piling up, waiting for possible use or final disposal. To date, the recovery of the residual 235U isotope contained in DU has been conducted only marginally by physical processes. Relative isotope abundances are often mediated by biological processes, and the biologically driven U isotopic fractionation has been previously identified in reducing bacteria. Our results indicate that the cells of two microalgal strains (freshwater Chlamydomonas sp. (ChlGS) and marine Tetraselmis mediterranea (TmmRU)) took up DU from the exposure solutions, inducing U isotopic fractionation with a preference for the fissile 235U isotope over 238U. The n(235U)/n(238U) isotopic fractionation magnitudes (δ235) were 23.6 ± 12.5‰ and 370.4 ± 103.9‰, respectively. These results open up new perspectives on the re-enrichment of DU tailings, offering a potential biological alternative to obtain reprocessed natural-equivalent uranium. Additionally, the findings present implications for identifying biological signatures in the geologic records.

Optimal strategies for bioremediation of nitrate-contaminated groundwater and microalgae biomass production.

Optimizing the mono-cultivation and mixed cultivation of Chlamydomonas reinhardtii, Chlorella vulgaris, and an Ettlia sp. was evaluated for treating nitrate-contaminated groundwater and biomass production. Ettlia sp. showed the highest nutrient assimilation and growth rate among the three microalgae during bioremediation. Light-dark cycle was the effective condition for nutrient removal and COD mitigation by microalgae. Mixed microalgae with a larger presence of the Ettlia sp. exhibited the highest biomass productivity, nitrate-nitrogen, and phosphate-phosphorus removal rates of 0.21 g/L/d, 16.6, and 3.06 mg/L/d, respectively. An N:P mass ratio of 5 was necessary to increase the mixed-microalgal performance. The settling efficiency of the mixed microalgae increased up to 0.55 when using pH modulation during 30 min. Therefore, applying an Ettlia sp.-dominant consortium was the optimum strategy for the bioremediation of nitrate-contaminated groundwater in 3 days.

Decreased phosphorus incorporation explains the negative effect of high iron concentrations in the green microalga Chlamydomonas acidophila.

The green microalga Chlamydomonas acidophila is an important primary producer in very acidic lakes (pH 2.0-3.5), characterized by high concentrations of ferric iron (up to 1 g total Fe L-1) and low rates of primary production. It was previously suggested that these high iron concentrations result in high iron accumulation and inhibit photosynthesis in C. acidophila. To test this, the alga was grown in sterilized lake water and in medium with varying total iron concentrations under limiting and sufficient inorganic phosphorus (Pi) supply, because Pi is an important growth limiting nutrient in acidic waters. Photosynthesis and growth of C. acidophila as measured over 5 days were largely unaffected by high total iron concentrations and only decreased if free ionic Fe3+ concentrations exceeded 100 mg Fe3+ L-1. Although C. acidophila was relatively rich in iron (up to 5 mmol Fe: mol C), we found no evidence of iron toxicity. In contrast, a concentration of 260 mg total Fe L-1 (i.e. 15 mg free ionic Fe3+ L-1), which is common in many acidic lakes, reduced Pi-incorporation by 50% and will result in Pi-limited photosynthesis. The resulting Pi-limitation present at high iron and Pi concentrations was illustrated by elevated maximum Pi-uptake rates. No direct toxic effects of high iron were found, but unfavourable chemical Pi-speciation reduced growth of the acidophile alga.

Optimization of triacylglycerol and starch production in Chlamydomonas debaryana NIES-2212 with regard to light intensity and CO2 concentration.

Triacylglycerol (TAG) and starch produced by micro-algae are potential sources of biofuel. Our previous studies showed that the unicellular green alga, Chlamydomonas debaryana NIES-2212, which is a rare species of Chlamydomonas that possesses phosphatidylcholine (PC), is a seed organism for the development of biofuel producers. This alga accumulates large amounts of TAG and starch under completely photo-autotrophic conditions during stationary phase without nutrient deprivation. The present study was performed to optimize the growth conditions of this alga with regard to light intensity and CO2 concentration to improve the efficiency of TAG and starch production. The growth rate of C. debaryana was greater at higher light intensity, although there was no significant difference in the final cell density of the culture. The highest contents of TAG and starch, approximately 200 fmol cell-1 and 600 pg cell-1, respectively, were achieved with a light intensity of 200 µmol m-2 s-1 bubbled with air containing 5.0 % CO2. These results suggest that optimization of light intensity and CO2 concentration can enhance the productivity of TAG and starch by C. debaryana NIES-2212.

Enhancement of total lipid yield by nitrogen, carbon, and iron supplementation in isolated microalgae.

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG-11 containing sodium carbonate concentration at 0.03 g · L-1 , and in normal BG-11 containing iron concentration (IC) at 0.009 or 0.012 g · L-1 . Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L-1 under the IC of 0.012 g · L-1 . Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties.

Anti-inflammatory effects of an oxylipin-containing lyophilised biomass from a microalga in a murine recurrent colitis model.

Diet and nutritional factors have emerged as possible interventions for inflammatory bowel diseases (IBD), which are characterised by chronic uncontrolled inflammation of the intestinal mucosa. Microalgal species are a promising source of n-3 PUFA and derived oxylipins, which are lipid mediators with a key role in the resolution of inflammation. The aim of the present study was to investigate the effects of an oxylipin-containing lyophilised biomass from Chlamydomonas debaryana on a recurrent 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis mice model. Moderate chronic inflammation of the colon was induced in BALB/c mice by weekly intracolonic instillations of low dose of TNBS. Administration of the lyophilised microalgal biomass started 2 weeks before colitis induction and was continued throughout colitis development. Mice were killed 48 h after the last TNBS challenge. Oral administration of the microalgal biomass reduced TNBS-induced intestinal inflammation, evidenced by an inhibition of body weight loss, an improvement in colon morphology and a decrease in pro-inflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-17. This product also down-regulated colonic expressions of inducible nitric oxide, cyclo-oxygenase 2 and NF-κB, as well as increased PPAR-γ. In addition, lyophilised microalgal biomass up-regulated the expressions of the antioxidant transcription factor nuclear factor E2-related factor 2 and the target gene heme oxygenase 1. This study describes for the first time the prophylactic effects of an oxylipin-containing lyophilised microalgae biomass from C. debaryana in the acute phase of a recurrent TNBS-induced colitis model in mice. These findings suggest the potential use of this microalga, or derived oxylipins, as a nutraceutical in the treatment of IBD.

Bioremediation of domestic and industrial wastewaters integrated with enhanced biodiesel production using novel oleaginous microalgae.

The study illustrates the synergistic potential of novel microalgal, Chlamydomonas debaryana IITRIND3, for phycoremediation of domestic, sewage, paper mill and dairy wastewaters and then subsequent utilisation of its biomass for biodiesel production. Among these wastewaters, maximum lipid productivity (87.5 ± 2.3 mg L-1 day-1) was obtained in dairy wastewater with removal efficiency of total nitrogen, total phosphorous, chemical oxygen demand and total organic carbon to be 87.56, 82.17, 78.57 and 85.97 %, respectively. Metal ions such as sodium, calcium, potassium and magnesium were also removed efficiently from the wastewaters tested. Pigment analysis revealed loss of chlorophyll a while increase in carotenoid content in algal cells cultivated in different wastewaters. Biochemical data of microalgae grown in different wastewaters showed reduction in protein content with an increase in carbohydrate and lipid contents. The major fatty acids in algal cells grown in dairy wastewater were C14:0, C16:0, C16:1, C18:0, C18:2 and C18:3. The physical properties of biodiesel derived from microalgae grown in dairy wastewater were in compliance with the ASTM D6751 and EN 14214 fuel standards and were comparable to plant oil methyl esters.

Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Hyperosmosis and its combination with nutrient-limitation are novel environmental stressors for induction of triacylglycerol accumulation in cells of Chlorella kessleri.

Triacylglycerols of oleaginous algae are promising for production of food oils and biodiesel fuel. Air-drying of cells induces triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, therefore, it seems that dehydration, i.e., intracellular hyperosmosis, and/or nutrient-limitation are key stressors. We explored this possibility in liquid-culturing C. kessleri cells. Strong hyperosmosis with 0.9 M sorbitol or 0.45 M NaCl for two days caused cells to increase the triacylglycerol content in total lipids from 1.5 to 48.5 and 75.3 mol%, respectively, on a fatty acid basis, whereas nutrient-limitation caused its accumulation to 41.4 mol%. Even weak hyperosmosis with 0.3 M sorbitol or 0.15 M NaCl, when nutrient-limitation was simultaneously imposed, induced triacylglycerol accumulation to 61.9 and 65.7 mol%, respectively. Furthermore, culturing in three-fold diluted seawater, the chemical composition of which resembled that of the medium for the combinatory stress, enabled the cells to accumulate triacylglycerol up to 24.7 weight% of dry cells in only three days. Consequently, it was found that hyperosmosis is a novel stressor for triacylglycerol accumulation, and that weak hyperosmosis, together with nutrient-limitation, exerts a strong stimulating effect on triacylglycerol accumulation. A similar combinatory stress would contribute to the triacylglycerol accumulation in air-dried C. kessleri cells.

Cytotoxic Activity of Microalgal-derived Oxylipins against Human Cancer Cell lines and their Impact on ATP Levels.

Oxylipins are metabolites derived from lipid peroxidation. The plant oxylipin methyl jasmonate (MJ) shows cytotoxic activity against cancer cell lines of various origins, with ATP-depletion being one of the mechanisms responsible for this effect. The cytotoxic activity of oxylipins (OXLs) isolated from the microalgae Chlamydomonas debaryana (13-HOTE) and Nannochloropsis gaditana (15-HEPE) was higher against UACC-62 (melanoma) than towards HT-29 (colon adenocarcinoma) cells. OXLs lowered the ATP levels of HT-29 and UACC-62 cells, but the effect was higher on the second cell line, which had higher basal ATP. This result proves a link between the cytotoxicity and the capability of these compounds to deplete ATP. In addition, the combination of 13-HOTE with the anticancer drug 5-fluorouracil (5-FU) induced a synergistic toxicity against HT-29 cells. These results highlight the therapeutic potential of oxylipins derived from microalgae.

Treating Urban Wastewater: Nutrient Removal by Using Immobilized Green Algae in Batch Cultures.

Essential roles of microalgae during the tertiary treatment of municipal wastewater have been proven. In order to avoid wash out of the biomass and also modify the quality of the treated wastewater; some techniques such as cell immobilization have been developed. To do so, in this study four samples from two species of microalgae (Chlorella vulgaris and Chlamydomonas sp.) were determined and confirmed by taxonomic identification. The samples were immobilized in calcium alginate beads. Within 10 days the amounts of nitrate (No3(-)-N) and orthophosphate (Po4(3-)-P) were measured to calculate the removal efficacy for each individual sample. Based on the standard methods, the amount of nitrate and orthophosphate were determined every 3 days within 10 days. The results indicate that immobilized microalgae are able to remove the nutrients and reduce the amount of nitrate and orthophosphate, significantly. Furthermore, the C. vulgaris (YG02) was the best species in this experience with 72% and 99% of reduction in the amount of nitrate and orthophosphate, respectively.