Nitrogen-depleted Conditions Research Articles

A New Species of Freshwater Algae Nephrochlamys yushanlensis sp. nov. (Selenastraceae, Sphaeropleales) and Its Lipid Accumulation during Nitrogen and Phosphorus Starvation

The new species Nephrochlamys yushanlensis sp. nov. is described from a freshwater plankton sample. A comparison of morphology, 18S rDNA gene and ITS2 sequences, and fatty acid profiles showed that the novel strain represents a new lineage within the genus Nephrochlamys. For the first time with a member of the Selenastraceae, experiments with phosphate and nitrate deprivation were conducted to evaluate changes in biomass, lipid and triacylglycerol (TAGs) accumulation, and composition of fatty acids. Biomass dry weight under simultaneous nitrogen and phosphorus depletion was 1.73g·L-1 , which is significantly lower than the 2.41g·L-1 observed in the control. All conditions of nutrient restriction significantly increased the lipid content in comparison with the control. The largest increase in the total lipid content, reaching 58.64% DW per cell at the end of cultivation, occurred with nitrogen deficiency. Significant increases in TAGs content (to 23.69% and 21.74%, respectively) occurred in phosphorus- and nitrogen-depleted conditions in comparison to the control (16.90%). Oleic (49.8-64.1%), palmitic (21.1-22.7%), and linoleic (8.6-10.3%) acids were the dominant fatty acids when cultured on standard BBM medium, as well as with the shortage of nutrients. Phosphorus deprivation as well as absence of both nitrogen and phosphorus led to the appearance of FAMEs α-linolenic (1.5-4.1%) and stearidonic (1.0-1.8%) acids. In general, FAME profiles revealed that the relative percentage of saturated and monounsaturated fatty acids increased (88.9% of total fatty acids) in nitrogen-depletion conditions, suggesting this strain may be suitable for biodiesel production.

Pulsed Electric Field Treatment Promotes Lipid Extraction on Fresh Oleaginous Yeast Saitozyma podzolica DSM 27192.

This study reports on the use of pulsed electric field (PEF) as a pre-treatment step to enhance lipid extraction yield using extraction with ethanol-hexane blend on fresh oleaginous yeast Saitozyma podzolica. The yeasts were cultivated on nitrogen-depleted condition and had a lipid content of 26.4 ± 4.6% of dry weight. PEF-treatment was applied on the yeast suspension either directly after harvesting (unwashed route) or after a washing step (washed route) which induced a reduction of conductivity by a factor eight. In both cases, cell concentration was 20 g of biomass per liter of suspension. In the unwashed route, the lipid extraction efficiency increased from 7% (untreated) to 54% thanks to PEF-treatment. In case an additional washing step was added after PEF-treatment, up to 81% of the lipid content could be recovered. The washed route was even more efficient since lipid extraction yields increased from 26% (untreated) to 99% of total lipid. The energy input for the PEF-treatment never exceeded 150 kJ per liter of initial suspension. The best lipid recovery scenario was obtained using pulses of 1 μs, an electric field of 40 kV/cm and it required slightly less than 11 MJ/kgLIPID. This amount of energy can be further reduced by at least a factor five by optimizing the treatment and especially by increasing the concentration of the treated biomass. The process can be easily up-scaled and does not require any expensive handling of the biomass such as freezing or freeze-drying. These findings demonstrate the potential benefit of PEF-treatment in the downstream processing of oleaginous yeast. From a basic research point of view, the influence of conductivity on PEF energy requirements and extraction yields was examined, and results suggest a higher efficiency of PEF-treatment in terms of energy when treatment is performed at lower conductivity.

Effects of different abiotic stresses on carotenoid and fatty acid metabolism in the green microalga Dunaliella salina Y6

PurposeUnder different abiotic-stress conditions, the unicellular green microalga Dunaliella salina accumulates large amounts of carotenoids which are accompanied by fatty acid biosynthesis. Carotenoids and fatty acids both possess long carbon backbones; however, the relationship between carotenoid and fatty acid metabolism is controversial and remains poorly understood in microalgae.MethodsIn this study, we investigated the growth curves and the β-carotene, lutein, lipid, and fatty acid contents of D. salina Y6 grown under different abiotic-stress conditions, including high light, nitrogen depletion, and high salinity.ResultsBoth high-salinity and nitrogen-depleted conditions significantly inhibited cell growth. Nitrogen depletion significantly induced β-carotene accumulation, whereas lutein production was promoted by high light. The accumulation of lipids did not directly positive correlate with β-carotene and lutein accumulation under the three tested abiotic-stress conditions, and levels of only a few fatty acids were increased under specific conditions.ConclusionOur data indicate that cellular β-carotene accumulation in D. salina Y6 positive correlates with accumulation of specific fatty acids (C16:0, C18:3n3, C14:0, and C15:0) rather than with total fatty acid content under different abiotic stress conditions.

Potassium channel KCN11 is required for maintaining cellular osmolarity during nitrogen starvation to control proper cell physiology and TAG accumulation in Chlamydomonas reinhardtii

BackgroundNitrogen (N) starvation in algae induces a variety of structural and metabolic changes including accumulation of triacylglycerol (TAG). Given the promising prospect of using algae as feedstock for biofuel production, accumulation of TAG upon N starvation becomes an ideal system to study TAG biosynthesis. Under nitrogen-depleted conditions, algae also accumulate compatible solutes such as sugar and certain amino acids, which is expected to elevate osmolarity in the cytoplasm. However, how osmoregulation is maintained and how it impacts on carbon metabolism, especially TAG accumulation under N starvation, are not well understood.ResultsWe show here that potassium channel KCN11 localized in the contractile vacuole (CV) mediates osmoregulation during N starvation and loss of KCN11 profoundly affects cell physiology and TAG biosynthesis. KCN11 level is increased and the CV pulsation is accelerated. Loss of KCN11 induces aberrant CV cycle, inhibition of cell growth, increase of cell size, inhibition of chlorophyll loss and TAG accumulation. These effects are rescued by addition of sucrose to raise osmolarity in the culture medium, indicating that osmoregulation is required for cell adaptation to N starvation. Metabolomic analysis shows reduction of acetyl-CoA and accumulation of glyceraldehyde-3-phosphate in kcn11 mutant relative to the control under N starvation, indicating that defects in acetyl-CoA biosynthesis and some metabolic steps from glyceraldehyde-3-phosphate to TAG contribute to the decreased TAG accumulation due to loss of osmoregulation.ConclusionsThis work provides novel insight of osmoregulation during N starvation in the control of cell physiology and metabolism especially TAG accumulation. According to these findings, we propose that osmolarity should be carefully monitored during the industrial production of biodiesel.

A method for the preparation of electrocompetent cells to transform unicellular green algae, Coccomyxa (Trebouxiophyceae, Chlorophyta) strains Obi and KJ

Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm−1 to the autospores yielded transformants at a frequency of >10−6 per input cell for strain KJ and >10−7 per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.

Nitrogen repletion favors cellular metabolism and improves eicosapentaenoic acid production in the marine microalga Isochrysis sp. CASA CC 101

Isochrysis sp. is a marine microalga and is a potential source of eicosapentaenoic acid and docosahexaenoic acid, which possess several health benefits. In general, Isochrysis sp. grow well in nitrate-rich culture medium which influences fatty acid biosynthesis; therefore, this study focused on how nitrate influences cellular metabolism in the marine microalga Isochrysis sp. CASA CC 101. Initially, response surface methodology was performed to optimize the level of major nutrients: nitrate and phosphate. The results showed that Isochrysis sp. when grown with nitrate at a concentration of 593 mg.L−1 produced higher eicosapentaenoic acid (7.3 mg.L1), whereas Isochrysis sp. when grown with higher concentration of phosphate did not produce higher levels of eicosapentaenoic acid. The influence of nitrate on cellular metabolism was further confirmed by culturing Isochrysis sp. at higher nitrate conditions. The cellular metabolic activity was analyzed by reactive oxygen species staining assay and the results showed that the cells exhibited reduced oxidative stress with lower green fluorescence under nitrate-repleted conditions. Furthermore, live/dead staining and cell viability assay revealed that live cell population was higher in nitrate-repleted conditions. Nile red and scanning electron microscopy analysis revealed that the cell size and intracellular lipid droplets of microalgal cells grown under nitrate-repleted cells were low, whereas the cell size and intracellular lipid droplets of the microalgal cells grown under nitrogen-depleted conditions were high. Further, the fatty acid profile of Isochrysis sp. grown under nitrogen-repleted conditions showed an increased eicosapentaenoic acid (22.52 mg.g−1 biomass). The mRNA expression of polyunsaturated fatty acid biosynthetic genes such as Δ5-desaturase (Δ5Des) was significantly elevated in nitrate-repleted conditions, whereas the expression of the Δ6-desaturase (Δ6Des) remained unaltered under all the conditions. This is the first study to demonstrate the correlation between nitrate repletion and cellular metabolic activity in terms of eicosapentaenoic acid production in Isochrysis sp. CASA CC 101.

The small Ca2+-binding protein CSE links Ca2+ signalling with nitrogen metabolism and filament integrity in Anabaena sp. PCC 7120

BackgroundFilamentous cyanobacteria represent model organisms for investigating multicellularity. For many species, nitrogen-fixing heterocysts are formed from photosynthetic vegetative cells under nitrogen limitation. Intracellular Ca2+ has been implicated in the highly regulated process of heterocyst differentiation but its role remains unclear. Ca2+ is known to operate more broadly in metabolic signalling in cyanobacteria, although the signalling mechanisms are virtually unknown. A Ca2+-binding protein called the Ca2+ Sensor EF-hand (CSE) is found almost exclusively in filamentous cyanobacteria. Expression of asr1131 encoding the CSE protein in Anabaena sp. PCC 7120 was strongly induced by low CO2 conditions, and rapidly downregulated during nitrogen step-down. A previous study suggests a role for CSE and Ca2+ in regulation of photosynthetic activity in response to changes in carbon and nitrogen availability.ResultsIn the current study, a mutant Anabaena sp. PCC 7120 strain lacking asr1131 (Δcse) was highly prone to filament fragmentation, leading to a striking phenotype of very short filaments and poor growth under nitrogen-depleted conditions. Transcriptomics analysis under nitrogen-replete conditions revealed that genes involved in heterocyst differentiation and function were downregulated in Δcse, while heterocyst inhibitors were upregulated, compared to the wild-type.ConclusionsThese results indicate that CSE is required for filament integrity and for proper differentiation and function of heterocysts upon changes in the cellular carbon/nitrogen balance. A role for CSE in transmitting Ca2+ signals during the first response to changes in metabolic homeostasis is discussed.

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

PurposeAnabaena sp. PCC7120 is a genetically tractable model organism for nitrogen fixation and photosynthesis research. The importance of small regulatory RNAs (sRNAs) as mediators of a number of cellular processes in bacteria has begun to be recognized. Bacterial sRNA binds to target genes through base pairing, and play a regulatory role. Many studies have shown that bacterial sRNA can regulate cell stress response, carbon and nitrogen fixation, and so on. However, little is known about sRNAs in Anabaena sp. PCC 7120 regarded to nitrogen fixation under later steady state.MethodsTo provide a comprehensive study of sRNAs in this model organism, the sRNA (< 200 nt) extracted from Anabaena sp. PCC 7120 under nitrogen step-down treatment of 12 days, together with the sRNA from the control, was analyzed using deep RNA sequencing. Possible target genes regulated by all identified putative sRNAs were predicted by IntaRNA and further analyzed for functional categorizations for biological pathways.ResultTotally, 14,132 transcripts were produced from the de novo assembly. Among them, transcripts that are located either in the intergenic region or antisense strand were kept, which resulted in 1219 sRNA candidates, for further analysis. RPKM-based differential expression analysis showed that 418 sRNAs were significantly differentially expressed between the samples from control (nitrogen addition, N+) and nitrogen depletion, (N−). Among them, 303 sRNAs were significantly upregulated, whereas 115 sRNAs were significantly downregulated. RT-PCR of 18 randomly chosen sRNAs showed a similar pattern as RNA-seq result, which confirmed the reliability of the RNA-seq data. In addition, the possible target genes regulated by unique sRNAs of Anabaena sp. PCC 7120 under nitrogen addition (N+) condition or that under nitrogen depletion (N−) condition were analyzed for functional categorization and biological pathways, which provided the evidences that sRNAs were indeed involved in many different metabolic pathways.ConclusionThe information from the present study provides a valuable reference for understanding the sRNA-mediated regulation of the nitrogen fixation in Anabaena PCC 7120 under steady state conditions.

Overexpression of fructose-1,6-bisphosphate aldolase 1 enhances accumulation of fatty acids in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtti is a promising source of bioenergy and understanding its carbon metabolism is important. In this organism, carbon accumulation can be manipulated with nitrogen supply. Chlamydomonas reinhardtii is a mixotroph that can act as an autotroph or heterotroph, depending on conditions. Fructose-1,6-bisphosphate aldolases (FBAs) are important enzymes in energy pathways of algae; they convert fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Expression of both CrFBA1 and CrFBA2 was less under nitrogen-deplete conditions than under nitrogen-replete conditions in Chlamydomonas cells. To explore CrFBA1 function, CrFBA1-overexpressing Chlamydomonas lines were generated. Compared to wild-type, transformed cells grew more slowly in photoautotrophic conditions, but at the same rate in mixotrophic condition. We suggest that overexpression of CrFBA1 affected photosynthetic carbon fixation in the Calvin-Benson cycle. The CrFBA1-overexpressing lines had less starch than the wild-type under mixotrophic, low-nitrogen conditions, but more fatty acids. In the transgenic cells, expression of granule bound starch synthase 1 (GBSS1) determined by quantitative reverse transcription-polymerase chain reaction was down-regulated, but acetyl-CoA synthetases (ACS1 and ACS2) and phosphoglucomutase (PGM) were up-regulated compared to wild-type. We propose three possible explanations for the observed low starch and high fatty acid content of the transgenic cells. Firstly, abnormal carbon flux by CrFBA1 overexpression leads to GBSS1 down-regulation in a feed-forward regulation, which resulted in the low starch accumulation. Secondly, increase of exogenous acetate assimilation by ACS1 and ACS2 up-regulation results in high accumulation of total fatty acid in transformants, perhaps due to compensation for reduced-carbon fixation. Lastly, activation of glycolytic pathway by CrFBA1 overexpression enhances conversion of starch to fatty acid. We conclude that overexpression of CrFBA1 deregulates carbon metabolism in Chlamydomonas reinhardtii.

Selective fractionation of free glucose and starch from microalgae using aqueous two-phase systems

Microalgae are a promising source of lipids, pigments, proteins and carbohydrates, which are valuable compounds for many industries. However, optimal fractionation and valorization of all produced compounds is necessary to improve the economic viability of microalgae production. This paper aims to understand the fractionation of microalgae carbohydrates (free glucose and starch) in aqueous two-phase systems. Three aqueous two-phase systems were investigated to efficiently and mildly separate carbohydrates from disrupted Neochloris oleoabundans. This strain contains 16 w/w% of proteins, 48 w/w% total fatty acids and 27 w/w% carbohydrates when cultivated under saline water and nitrogen depletion conditions. The protein content decreases and the amount of fatty acids and carbohydrates increases notably under stress conditions and glucose becomes the main carbohydrate in this microalgae. Glucose is present in the disrupted microalgae as part of polymeric carbohydrates (starch) or in monomeric form (free glucose). With the aqueous two-phase system Polyethylene Glycol 400 - Cholinium dihydrogen phosphate (PEG400-ChDHp) microalgal free glucose is fractionated up to a recovery of 99% to the most hydrated bottom phase in a single step. Simultaneously, a recovery of 70% is reached for microalgal starch in the interface after two additional liquid-liquid extractions with PEG400-ChDHp. The final fractions obtained were free of pigments.

Characterization of Chlamydomonas Very High Light-tolerant Mutants for Enhanced Lipid Production.

Biodiesel production from microalgae is still not commercially realized due to the high cost of production. High light-tolerance has been suggested as a desirable phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Nevertheless, it has not been shown if algae with such a phenotype would have better efficiency for lipid production. To determine lipid productivity in high light-tolerant mutants, and to understand the pathways involved in high light-tolerant phenotype, two very high light-tolerant mutants of the green alga Chlamydomonas reinhardtii - CAL028_01_28 and CAL034_01_48 - were selected from eighteen high light-tolerant mutants from the CAL collection. Under high light intensity conditions, and the presence of reactive oxygen species, which are conditions constantly experienced by algae growing in open-pond environments, these strains exhibited higher photosynthetic efficiency and improved survival. The physiological characterization of these mutants revealed that the detoxification of ROS by carotenoids and antioxidant enzymes is crucial for their growth under high light conditions. Neither mutant was affected in terms of its ability to accumulate lipid under nitrogen-depleted condition. More importantly, lipid productivity under high light conditions increased twofold in these mutants compared to that of the wild-type. Taken together, very high light-tolerant mutants confer a high potential for biofuel production under outdoor conditions, and their improved ability to survive under oxidative stress is an important key for efficient growth under outdoor conditions.

Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker’s yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol–producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl–CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol–producing enzyme fatty acyl–CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

Strategies among phytoplankton in response to alleviation of nutrient stress in a subtropical gyre.

Despite generally low primary productivity and diatom abundances in oligotrophic subtropical gyres, the North Atlantic Subtropical Gyre (NASG) exhibits significant diatom-driven carbon export on an annual basis. Subsurface pulses of nutrients likely fuel brief episodes of diatom growth, but the exact mechanisms utilized by diatoms in response to these nutrient injections remain understudied within near-natural settings. Here we simulated delivery of subsurface nutrients and compare the response among eukaryotic phytoplankton using a combination of physiological techniques and metatranscriptomics. We show that eukaryotic phytoplankton groups exhibit differing levels of transcriptional responsiveness and expression of orthologous genes in response to release from nutrient limitation. In particular, strategies for use of newly delivered nutrients are distinct among phytoplankton groups. Diatoms channel new nitrate to growth-related strategies while physiological measurements and gene expression patterns of other groups suggest alternative strategies. The gene expression patterns displayed here provide insights into the cellular mechanisms that underlie diatom subsistence during chronic nitrogen-depleted conditions and growth upon nutrient delivery that can enhance carbon export from the surface ocean.

Complete genome sequence of Nitratireductor sp. strain OM-1: A lipid-producing bacterium with potential use in wastewater treatment

Reducing CO2 emissions is necessary to alleviate rising global temperature. Renewable sources of energy are becoming an increasingly important substitute for fossil fuels. An important step in this direction is the isolation of novel, technologically relevant microorganisms. Nitratireductor sp. strain OM-1 can convert volatile short-chain fatty acids in wastewater into 2-butenoic acid and its ester and can accumulate intracellularly esterified compounds up to 50% of its dried cell weight under nitrogen-depleted conditions. It is believed that a novel fatty acid biosynthesis pathway including an esterifying enzyme is encoded in its genome. In this study, we report the whole-genome sequence (4.8 Mb) of OM-1, which comprises a chromosome (3,977,827 bp) and a megaplasmid (857,937 bp). This sequence information provides insight into the genome organization and biochemical pathways of OM-1. In addition, we identified lipid biosynthesis pathways in OM-1, paving the way to a better understanding of its biochemical characterization.

Dissecting lipid accumulation of microalgae Nannochloropsis oculata using fluorescent image analysis

Cell suspension of Nannochloropsis oculata was cultured in a modified f/2 medium to study the changes of lipid content during phases of growth. The growth of cell suspension was determined by measuring the cell density and diameter under light microscope. To observe and evaluate the accumulation of lipid droplets in microalgae cells, lipid droplets were stained with Nile Red fluorescent dye then examined under fluorescence microscope and the obtained images were analyzed using Fiji ImageJ, an image processing program. The cell density increased quickly at the first 6 days of culture while cell diameter reached the highest value at the 8th day and 20th day of culture. The presence of lipid droplets in the cells could be observed from the 20th day of culture. The size of lipid droplets was gradually increased after 60 days. Treatment of depleted nitrogen for 4 days resulted an increase in the accumulation of lipid. The intracellular lipid accumulation during phases of growth of the cell suspension under nitrogen-depleted conditions was also discussed.

Evaluation of Marine Synechococcus for an Algal Biorefinery in Arid Regions

Implementing microalgae biorefinery in arid environments requires utilization of strains that can grow at high temperatures (above 28 °C) and salinity levels (above 30 ppt). In this study, we investigate the newly isolated seawater strain, Synechococcus, native to the United Arab Emirates, and evaluate its value as a perspective organism for cultivation (for fuel and bio-products) in regions with freshwater scarcity. The strain displayed tolerance to a wide range of temperature (22–37 °C) and salinity (20–41 ppt), with maximum biomass concentration of 0.72 g L−1 and a maximum growth rate of 82 mg L−1 d−1 at 25 °C and 33 ppt salinity. Lipids accumulation reached up to 26% of dry weight in nitrogen-depleted conditions (with 1.8 mM of nitrates addition to the media), whereas protein content exceeded 50% dry weight. In this study, harvesting is investigated using three chemical agents: Ferric chloride, sodium hydroxide, and chitosan. Cell disruption is analyzed for four distinct treatments: Enzymatic, alkaline, ultrasonic, and hydrothermal. Among tested methods, flocculation with sodium hydroxide and ultrasonication were found to be the most efficient techniques for harvesting and cell disruption, respectively. The growth characteristics of the local strain and the potential to derive protein and lipids from it makes it a promising biomass in a biorefinery context.

Monitoring of the Photosynthetic Activity of the Microalgae Chlorella under Nitrogen Depletion Conditions

Abstract—The paper presents the results of monitoring of the photosynthetic activity of the green microalga Chlorella. A thermophilic strain of Chlorella with a high rate of cell division was cultivated in a photobioreactor. Three medium variants with different mineral composition were used. Chlorophyll fluorescence induction curves and microalgal suspension absorption spectra were recorded every hour during 40-hour culture growth for each medium. The method of spectral multiexponential approximation we have developed was used to analyze 120 induction curves obtained after monitoring. This method revealed the dynamics of gradual changes in the number of phases of induction curves, their amplitudes and characteristic times during culture growth. The relationship between the parameters of induction curves and electron transfer processes in the reaction center of photosystem II allowed for interpretation of the changes in the shape of the induction curves in terms of the processes in the reaction center of photosystem II. The spectral multiexponential approximation method was shown to be highly sensitive to physiological state of microalgae and suitable for analyzing a huge amount of experimental data. The physiological processes accompanying the depletion of nitrogen in the environment are discussed.

Comparative transcriptome analysis of a long-time span two-step culture process reveals a potential mechanism for astaxanthin and biomass hyper-accumulation in Haematococcus pluvialis JNU35

BackgroundAmong all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process.ResultsIn the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass.ConclusionsTo the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

The NnaR orphan response regulator is essential for the utilization of nitrate and nitrite as sole nitrogen sources in mycobacteria

Nitrogen is an essential component of biological molecules and an indispensable microelement required for the growth of cells. Nitrogen metabolism of Mycobacterium smegmatis is regulated by a number of transcription factors, with the glnR gene product playing a major role. Under nitrogen-depletion conditions, GlnR controls the expression of many genes involved in nitrogen assimilation, including the msmeg_0432 gene encoding NnaR, the homologue of a nitrite/nitrate transport regulator from Streptomyces coelicolor. In the present study, the role of NnaR in the nitrogen metabolism of M. smegmatis was evaluated. The ∆glnR and ∆nnaR mutant strains were generated and cultured under nitrogen-depletion conditions. Total RNA profiling was used to investigate the potential role of NnaR in the GlnR regulon under nitrogen-depletion and in nitrogen-rich media. We found that disruption of MSMEG_0432 affected the expression of genes involved in nitrite/nitrate uptake, and its removal rendered mycobacteria unable to assimilate nitrogen from those sources, leading to cell death. RNA-Seq results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assays (EMSAs). The ability of mutants to grow on various nitrogen sources was evaluated using the BIOLOG Phenotype screening platform and confirmed on minimal Sauton’s medium containing various sources of nitrogen. The ∆glnR mutant was not able to convert nitrates to nitrites. Interestingly, NnaR required active GlnR to prevent nitrogen starvation, and both proteins cooperated in the regulation of gene expression associated with nitrate/nitrite assimilation. The ∆nnaR mutant was able to convert nitrates to nitrites, but it could not assimilate the products of this conversion. Importantly, NnaR was the key regulator of the expression of the truncated haemoglobin trHbN, which is required to improve the survival of bacteria under nitrosative stress.

Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ

BackgroundOxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels > 60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired.ResultsIn this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5 ms) electric pulse at a high field strength (7500 V cm−1) followed by multiple 50-ms electric pulses at low field strength (250 V cm−1). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%.ConclusionsOur study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.