Search

BACKGROUND RESULTS CONCLUSION Nannochloropsis algae contain approximately 20% polyunsaturated fatty acids (PUFA) and hold significant potential for high‐quality eicosapentaenoic acid (EPA) food industrialization. However, EPA in Nannochloropsis sp. is prone to oxidation, and microbial growth is a critical factor affecting the shelf life of fresh food. Storage composition and temperature are primary factors influencing microbial growth, yet these aspects are not fully understood. This study investigates the effects of temperature and encapsulation on EPA content in nano‐products over time. Nano‐powder and nanobeads derived from Nannochloropsis sp. served as raw materials. Additionally, changes in aerobic plate counts and coliform groups were monitored.The results indicated that nanobeads, due to their more complex processing and less mature packaging, were more susceptible to coliform contamination compared to nano‐powder. In terms of EPA stability, nanobeads exhibited a longer storage life than nano‐powder. The oxidation rate of both nano‐powder and nanobeads was faster at 37 °C than at 25 °C.These findings can inform general shelf life estimation, rapid detection of total lipid content in nano‐products and macro extraction of nano‐oil. Moreover, they have significant implications for delaying EPA oxidation in nano‐products and improving hygienic quality control for microbial detection. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Microalgae often undergo different CO2 experiment in their habitat. To adapt to low CO2, carbon concentrating mechanism (CCM) could be launched in majority of microalgae and CCM are regulated at RNA level are well known. However, epigenetic modifications and their potential regulation of the transcription of masked genes at the genome level in response to CO2 fluctuation remain unclear. Here epigenetic regulation in response to CO2 fluctuation and epigenome-association with phenotypic plasticity of CCM are firstly uncovered in marine microalga Nannochloropsis oceanica IMET1. The result showed that lysine butyrylation (Kbu) and histone H3K9m2 modifications were present in N. oceanica IMET1. Moreover, Kbu modification positively regulated gene expression. In response to CO2 fluctuation, there were 5,438 and 1,106 genes regulated by Kbu and H3K9m2 in Nannochloropsis, respectively. Gained or lost histone methylations were closely associated with activating or repressing gene expressions. Differential modifications were mainly enriched in carbon fixation, photorespiration, photosynthesis, and lipid metabolism etc. Massive genome-wide epigenetic reprogramming was observed after N. oceanica cells shifted from high CO2 to low CO2. Particularly, we firstly noted that the transcription of the key low CO2 responsive carbonic anhydrase (CA5), a key component involved in CCM stress signaling, was potentially regulated by bivalent Kbu-H3K9m2 modifications in microalgae. This study provides novel insights into the relationship between gene transcription and epigenetic modification in Nannochloropsis, which will lay foundation on genetic improvement of CCM at epigenetic level. [ABSTRACT FROM AUTHOR]

Abstract This study investigated the first-ever reported use of freshwater Nannochloropsis for the bioremediation of dairy processing side streams and co-generation of valuable products, such as β-galactosidase enzyme. In this study, N. limnetica was found to grow rapidly on both autoclaved and non-autoclaved whey-powder media (referred to dairy processing by-product or DPBP) without the need of salinity adjustment or nutrient additions, achieving a biomass concentration of 1.05–1.36 g L−1 after 8 days. The species secreted extracellular β-galactosidase (up to 40.84 ± 0.23 U L−1) in order to hydrolyse lactose in DPBP media into monosaccharides prior to absorption into biomass, demonstrating a mixotrophic pathway for lactose assimilation. The species was highly effective as a bioremediation agent, being able to remove > 80% of total nitrogen and phosphate in the DPBP medium within two days across all cultures. Population analysis using flow cytometry and multi-channel/multi-staining methods revealed that the culture grown on non-autoclaved medium contained a high initial bacterial load, comprising both contaminating bacteria in the medium and phycosphere bacteria associated with the microalgae. In both autoclaved and non-autoclaved DPBP media, Nannochloropsis cells were able to establish a stable microalgae–bacteria interaction, suppressing bacterial takeover and emerging as dominant population (53–80% of total cells) in the cultures. The extent of microalgal dominance, however, was less prominent in the non-autoclaved media. High initial bacterial loads in these cultures had mixed effects on microalgal performance, promoting β-galactosidase synthesis on the one hand while competing for nutrients and retarding microalgal growth on the other. These results alluded to the need of effective pre-treatment step to manage bacterial population in microalgal cultures on DPBP. Overall, N. limnetica cultures displayed competitive β-galactosidase productivity and propensity for efficient nutrient removal on DPBP medium, demonstrating their promising nature for use in the valorisation of dairy side streams.

The current study focused on natural breeding techniques using locally available mud crab broods during breeding season. The breeding trial was tested in artificially created earthen pens that designed with mangrove plants and salt tolerance grass. Mature brood crabs were collected from local rivers and equally distributed in 20 L black color plastic containers in three different salinities (T1 = 24ppt, T2 = 26ppt, and T3 = 28ppt) and covered with black cloth. The river salinity was 26ppt during breeding season that is considered as control(T2), and treatment T1 and T3 were prepared artificially mixing with water and sea salt respectively. Broods are reared in containers until hatching and zoea was transferred to earthen pens immediately after hatching. Zoea were reared in earthen pens until turns to crablet(0.027 ± 0.005 g). The broods as well as crablet were identified through molecular barcoding and confirmed species as Scylla olivicea. Besides, morphometric characters like weight(126.22 ± 0.19 g), carapace length(6.36 ± 0.08 cm), carapace width(9.08 ± 0.08 cm), carapace color(brownish to brownish green), Anterolateral margin and teeth (3.79 ± 0.05 cm), abdomen length(3.04 ± 0.02 cm), hand (5.50 ± 0.03 cm), carpus(2.54 ± 0.04), antenna (2.69 ± 0.07), frontal margin(2.90 ± 0.07 cm), dactylus(3.13 ± 0.06 cm), thoracic sternites(4.17 ± 0.10), merus(4.16 ± 0.11 cm) etc. are also assessed during the experiment. The hatching rate, survival %, incubation period and other parameters are significantly different among the treatments. The highest hatching rate was observed in T2(91.34 ± 4.61 %) and lowest was in T1(57.98 ± 1.38) whereas the highest survival rate was recorded in T2(10.53 ± 1.56) and lowest observed in T1(2.87 ± 0.68). The order of incubation was T1(12.3 days) > T3(11 days) > T2(8.67days) observed during experiment. The cannibalism rate was also estimated in this experiment and observed cannibalism rate was 71.20 ± 2.08. Natural mud crab breeding is easy and can be applicable in fields at low cost with significant survival rate. [ABSTRACT FROM AUTHOR]

Microalgae have rigid, complex cell walls hindering direct lipid extraction. Cell disruption techniques are used to rupture these cellular structures to increase lipid extraction. Researchers investigating the downstreamprocessing of microalgae do not always have access to microalgal cultivation systems to generate large amounts of fresh microalgal biomass. Using resuspended freeze-dried microalgal biomass as a model laboratory system for concentrated fresh biomass during cell disruption experiments offers greater flexibility in experimental planning and omits investment costs of microalgal cultivation equipment. So far, it however remains unclear whether freeze-dried resuspended biomass can be used as a model laboratory system to represent concentrated fresh biomass during cell disruption and lipid extraction experiments. This paper thus evaluated the suitability of resuspended freeze-dried Nannochloropsis as a model laboratory system for concentrated fresh Nannochloropsis during cell disruption. Ultrasound assisted cell disruption was used as example cell disruption technique and lipid extraction efficiency and free fatty acid content were investigated. Tap water and 3% sodium chloride are both suitable resuspension media for the resuspension of freeze-dried Nannochloropsis. Resuspension duration should be limited (< 120 min) to prevent the formation of free fatty acids. The condition of the biomass (concentrated fresh, or resuspended freeze-dried) prior to ultrasound assisted cell disruption did not influence the resulting lipid extraction efficiency. Resuspended freeze-dried Nannochloropsis biomass in tap water or 3% sodium chloride can thus be used as a model laboratory system for fresh microalgal biomass during research on ultrasound assisted lipid extraction. The generalization of the results to other cultivation conditions, cell disruption techniques, components of interest or microalgal species should be carefully assessed. [ABSTRACT FROM AUTHOR]

Simple Summary: With the increase in the global population, food and energy production systems need to find immediately effective alternatives. There has been increased interest in microalgae thanks to their ability to grow exploiting photosynthesis with only water and simple nutrients, even derived from wastewater. Among microalgae, Nannochloropsis genus is one of the most interesting ones due to the capacity to accumulate "good" fatty acids, omega-3 and omega-6. Currently, the cultivation of microalgae is not often cost-effective, and strain domestication must be performed. Microalgae can be manipulated through metabolic engineering to increase their biomass productivity or to obtain high-value products that overcome the cultivation cost. Here, we review the main genetic manipulation tools and present some examples to explain the potential of Nannochloropsis spp. as a green cell bio-factory. The use of genetic engineering will finally bridge the gap between the probable and feasible use of microalgae. Securing food, energy, and raw materials for a growing population is one of the most significant challenges of our century. Algae play a central role as an alternative to plants. Wastewater and flue gas can secure nutrients and CO2 for carbon fixation. Unfortunately, algae domestication is necessary to enhance biomass production and reduce cultivation costs. Nannochloropsis spp. have increased in popularity among microalgae due to their ability to accumulate high amounts of lipids, including PUFAs. Recently, the interest in the use of Nannochloropsis spp. as a green bio-factory for producing high-value products increased proportionally to the advances of synthetic biology and genetic tools in these species. In this review, we summarized the state of the art of current nuclear genetic manipulation techniques and a few examples of their application. The industrial use of Nannochloropsis spp. has not been feasible yet, but genetic tools can finally lead to exploiting this full-of-potential microalga. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Arid Land Studies is the property of Japanese Association for Arid Land Studies and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Microalgae have emerged as a promising feedstock for the sustainable production of lipid-based products, including biofuels and omega-3 fatty acids, by utilizing CO2 as a carbon source. Glycerol-3-phosphate dehydrogenase (GPDH) is a vital enzyme in microalgal lipid synthesis, converting dihydroxyacetone phosphate into glycerol-3-phosphate, the initial substrate for the Kennedy pathway responsible for triacylglycerol production. In this study, we identified and overexpressed a chloroplastic NAD(P)+-dependent GPDH (NsGPDH1) in Nannochloropsis salina. Quantitative reverse transcription polymerase chain reaction and western blotting revealed increased NsGPDH1 expression in the transformants as compared to wild-type N.salina. This overexpression of NsGPDH1 resulted in increased glycerol production and a higher proportion of mono-unsaturated fatty acids, which are indicative of GPDH overexpression. Notably, lipid productivity in NsGPDH1-overexpressing N.salina strains increased by 40% without significant growth defects. These results highlight the effectiveness of NsGPDH1 overexpression as a strategy for enhancing lipid biosynthesis in N. salina, contributing to the advancement of microalgae-based lipid production. [ABSTRACT FROM AUTHOR]

In light of the escalating global energy crisis, microalgae have emerged as highly promising producers of biofuel and high-value products. Among these microalgae, Nannochloropsis has received significant attention due to its capacity to generate not only triacylglycerol (TAG) but also eicosapentaenoic acid (EPA) and valuable carotenoids. Recent advancements in genetic tools and the field of synthetic biology have revolutionized Nannochloropsis into a powerful biofactory. This comprehensive review provides an initial overview of the current state of cultivation and utilization of the Nannochloropsis genus. Subsequently, our review examines the metabolic pathways governing lipids and carotenoids, emphasizing strategies to enhance oil production and optimize carbon flux redirection toward target products. Additionally, we summarize the utilization of advanced genetic manipulation techniques in Nannochloropsis. Together, the insights presented in this review highlight the immense potential of Nannochloropsis as a valuable model for biofuels and synthetic biology. By effectively integrating genetic tools and metabolic engineering, the realization of this potential becomes increasingly feasible. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effects of dietary supplementation of Nannochloropsis on gene expression and serum metabolic profile in growing Barki lambs. For this reason, twenty apparently healthy growing Barki lambs were allocated into two equal groups (10 lambs each). Control group received the basal diet whereas the supplemented group received the basal diet with Nannochloropsis powder which given in the concentrate at a rate of 10 g /kg/day for subsequent 30 days. A blood sample was collected from each lamb via jugular venipuncture before starting the experiment (T0), (T15) and (T30) after supplementation for hematobiochemical examination and real time PCR. Gene’s expression of ACACA, FASN, SCD and FABP4 were significantly up-regulated in supplemented lambs at (T15) and (T30) while DGAT1, insulin, glucokinase and glucose transporter showed a significant up-regulation at day 30 compared to control ones. Supplemented lambs had a significant (P < 0.05) increase of final body weights, daily weight gain and feed intake with significant (P<0.05) decrease of feed conversion ratio. From the third week onwards, the body weight, body length and body condition score were significantly (P > 0.05) higher in the supplemented group. There was a significant (P<0.05) increase in Hb, RBC, WBC, serum level of glucose, insulin, triglyceride, total protein, globulin and BUN with significant (p<0.05) decrease of cholesterol, ALT and AST in supplemented lambs at T30. The results suggest that Nannochloropsis could be used as a useful supplement to improve health status and body growth of growing Barki lambs. [ABSTRACT FROM AUTHOR]

Microalgae have rigid, complex cell walls hindering direct lipid extraction. Cell disruption techniques are used to rupture these cellular structures to increase lipid extraction. Researchers investigating the downstream processing of microalgae do not always have access to microalgal cultivation systems to generate large amounts of fresh microalgal biomass. Using resuspended freeze-dried microalgal biomass as a model laboratory system for concentrated fresh biomass during cell disruption experiments offers greater flexibility in experimental planning and omits investment costs of microalgal cultivation equipment. So far, it however remains unclear whether freeze-dried resuspended biomass can be used as a model laboratory system to represent concentrated fresh biomass during cell disruption and lipid extraction experiments. This paper thus evaluated the suitability of resuspended freeze-dried Nannochloropsis as a model laboratory system for concentrated fresh Nannochloropsis during cell disruption. Ultrasound assisted cell disruption was used as example cell disruption technique and lipid extraction efficiency and free fatty acid content were investigated. Tap water and 3% sodium chloride are both suitable resuspension media for the resuspension of freeze-dried Nannochloropsis. Resuspension duration should be limited (< 120 min) to prevent the formation of free fatty acids. The condition of the biomass (concentrated fresh, or resuspended freeze-dried) prior to ultrasound assisted cell disruption did not influence the resulting lipid extraction efficiency. Resuspended freeze-dried Nannochloropsis biomass in tap water or 3% sodium chloride can thus be used as a model laboratory system for fresh microalgal biomass during research on ultrasound assisted lipid extraction. The generalization of the results to other cultivation conditions, cell disruption techniques, components of interest or microalgal species should be carefully assessed.

Abstract Background Genomic three-dimensional (3D) spatial organization plays a key role in shaping gene expression and associated chromatin modification, and it is highly sensitive to environmental stress conditions. In microalgae, exposure to nitrogen stress can drive lipid accumulation, yet the associated functional alterations in the spatial organization of the microalgal genome have yet to be effectively characterized. Results Accordingly, the present study employed RNA-seq, Hi-C, and ChIP-seq approaches to explore the relationship between 3D chromosomal architecture and gene expression during lipid accumulation in the marine microalga Nannochloropsis oceanica in response to nitrogen deprivation (ND). These analyses revealed that ND resulted in various changes in chromosomal organization, including A/B compartment transitions, topologically associating domain (TAD) shifts, and the disruption of short-range interactions. Significantly higher levels of gene expression were evident in A compartments and TAD boundary regions relative to B compartments and TAD interior regions, consistent with observed histone modification enrichment in these areas. ND-induced differentially expressed genes (DEGs) were notably enriched in altered TAD-associated regions and regions exhibiting differential genomic contact. These DEGs were subjected to Gene Ontology (GO) term analyses that indicated they were enriched in the ‘fatty acid metabolism’, ‘response to stress’, ‘carbon fixation’ and ‘photosynthesis’ functional categories, in line with the ND treatment conditions used to conduct this study. These data indicate that Nannochloropsis cells exhibit a clear association between chromatin organization and transcriptional activity under nitrogen stress conditions. Pronounced and extensive histone modifications were evident in response to ND. Observed changes in chromatin architecture were linked to shifts in histone modifications and gene expression. Conclusions Overall, the reprogramming of many lipid metabolism-associated genes was evident under nitrogen stress conditions with respect to both histone modifications and chromosomal organization. Together these results revealed that higher-order chromatin architecture represents a new layer that can guide efforts to understand the transcriptional regulation of lipid metabolism in nitrogen-deprived microalgae.

IntroductionAlmegaPL® is an oil rich in polar-lipid (> 15% w/w) derived from the microalga Nannochloropsis, that contains exclusively eicosapentaenoic acid (EPA > 25% w/w), without the DHA that is present in all other natural sources of omega-3. Previous findings from a randomized controlled clinical trial demonstrated the ability of AlmegaPL® supplementation to reduce cholesterol levels.MethodsIn this post-market cohort study, we built upon previous findings and targeted the actual end-users of the supplement. Participants were recruited from a new subscriber database of AlmegaPL® capsules (1000–1100 mg/day) to capture the complexity of real-world clinical and consumer settings. Changes in circulating triglycerides (TG), remnant cholesterol (RC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), total cholesterol (TC), high-sensitivity C-reactive protein (hs-CRP), glucose and glycated hemoglobin (HbA1c) were monitored at baseline, Month 3, and Month 6 of supplementation using the at-home Baseline Heart Health Testing Kit by Imaware® (Houston, TX, USA).ResultsParticipants, who had, on average, normal TG level at baseline (1.62 ± 0.60 mmol/L), experienced a significant and progressive decrease in TG at Month 3 (8.0%; −0.13 ± 0.59 mmol/L; p < 0.001) and Month 6 (14.2%; −0.23 ± 0.64 mmol/L; p < 0.001) (primary outcome). Furthermore, after 6 months of supplementation, TC and non-HDL-cholesterol decreased by 5.0% (−0.26 ± 0.98 mmol/L; p < 0.001) and 5.5% (−0.21 ± 0.86 mmol/L; p < 0.001) respectively, primarily driven by a 14.9% reduction in RC (−0.11 ± 0.29 mmol/L; p < 0.001).DiscussionConsistent with our previous clinical trial, the decrease in RC was not coupled to an increase in LDL, which seems to be a benefit associated with EPA-only based formulations. In addition, this study demonstrated the AlmegaPL® capacity to maintain already healthy TG levels by further inducing a 14.9% decrease. Collectively, these findings highlight AlmegaPL® uniqueness as a natural over-the-counter option for EPA-only polar lipid that appears particularly effective in maintaining blood lipid levels in a generally healthy, normolipidemic population.Clinical trial registrationhttps://clinicaltrials.gov/, identifier NCT05267301

Species in genus Nannochloropsis, especially N. oceanica and N. gaditana, have been evolving as the model microalgae for both application and theory studies. The position effect of genome integration, the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments. The molecular tools including genetic transformation, homologous recombination, gene edition, gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species. The construction of animal and plant artificial chromosomes with "top down" strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes (NannoACs). It seems that the methods and materials to set the foundation for constructing NannoACs are at hands. In this review, we outlined the current status of transgenes in Nannochloropsis species, summarized the limitations of both integrative and non-integrative vectors, and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first, and then truncating the natural chromosomes. NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds, thus contributing towards its genetic improvement and synthetic biological studies. [ABSTRACT FROM AUTHOR]

The cell cycle is the fundamental cellular process of eukaryotes. Although cell-cycle-related genes have been identified in microalgae, their cell cycle progression differs from species to species. Cell enlargement in microalgae is an essential biological trait. At the same time, there are various causes of cell enlargement, such as environmental factors, especially gene mutations. In this study, we first determined the phenotypic and biochemical characteristics of a previously obtained enlarged-cell-size mutant of Nannochloropsis oceanica, which was designated ECS. Whole-genome sequencing analysis of the insertion sites of ECS indicated that the insertion fragment is integrated inside the 5′-UTR of U/P-type cyclin CYCU;1 and significantly decreases the gene expression of this cyclin. In addition, the transcriptome showed that CYCU;1 is a highly expressed cyclin. Furthermore, cell cycle analysis and RT-qPCR of cell-cycle-related genes showed that ECS maintains a high proportion of 4C cells and a low proportion of 1C cells, and the expression level of CYCU;1 in wild-type (WT) cells is significantly increased at the end of the light phase and the beginning of the dark phase. This means that CYCU;1 is involved in cell division in the dark phase. Our results explain the reason for the larger ECS size. Mutation of CYCU;1 leads to the failure of ECS to fully complete cell division in the dark phase, resulting in an enlargement of the cell size and a decrease in cell density, which is helpful to understand the function of CYCU;1 in the Nannochloropsis cell cycle. [ABSTRACT FROM AUTHOR]

Securing food, energy, and raw materials for a growing population is one of the most significant challenges of our century. Algae play a central role as an alternative to plants. Wastewater and flue gas can secure nutrients and CO2 for carbon fixation. Unfortunately, algae domestication is necessary to enhance biomass production and reduce cultivation costs. Nannochloropsis spp. have increased in popularity among microalgae due to their ability to accumulate high amounts of lipids, including PUFAs. Recently, the interest in the use of Nannochloropsis spp. as a green bio-factory for producing high-value products increased proportionally to the advances of synthetic biology and genetic tools in these species. In this review, we summarized the state of the art of current nuclear genetic manipulation techniques and a few examples of their application. The industrial use of Nannochloropsis spp. has not been feasible yet, but genetic tools can finally lead to exploiting this full-of-potential microalga.

Background: Genomic three-dimensional (3D) spatial organization plays a key role in shaping gene expression and associated chromatin modification, and it is highly sensitive to environmental stress conditions. In microalgae, exposure to nitrogen stress can drive lipid accumulation, yet the associated functional alterations in the spatial organization of the microalgal genome have yet to be effectively characterized. Results: Accordingly, the present study employed RNA-seq, Hi-C, and ChIP-seq approaches to explore the relationship between 3D chromosomal architecture and gene expression during lipid accumulation in the marine microalga Nannochloropsis oceanica in response to nitrogen deprivation (ND). These analyses revealed that ND resulted in various changes in chromosomal organization, including A/B compartment transitions, topologically associating domain (TAD) shifts, and the disruption of short-range interactions. Significantly higher levels of gene expression were evident in A compartments and TAD boundary regions relative to B compartments and TAD interior regions, consistent with observed histone modification enrichment in these areas. ND-induced differentially expressed genes (DEGs) were notably enriched in altered TAD-associated regions and regions exhibiting differential genomic contact. These DEGs were subjected to Gene Ontology (GO) term analyses that indicated they were enriched in the 'fatty acid metabolism', 'response to stress', 'carbon fixation' and 'photosynthesis' functional categories, in line with the ND treatment conditions used to conduct this study. These data indicate that Nannochloropsis cells exhibit a clear association between chromatin organization and transcriptional activity under nitrogen stress conditions. Pronounced and extensive histone modifications were evident in response to ND. Observed changes in chromatin architecture were linked to shifts in histone modifications and gene expression. Conclusions: Overall, the reprogramming of many lipid metabolism-associated genes was evident under nitrogen stress conditions with respect to both histone modifications and chromosomal organization. Together these results revealed that higher-order chromatin architecture represents a new layer that can guide efforts to understand the transcriptional regulation of lipid metabolism in nitrogen-deprived microalgae. [ABSTRACT FROM AUTHOR]

The overall goal of this study was to provide solutions to innovative microalgaebased technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second "stress" phase using the RAS wastewater to enhance the production of highvalue metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and bcarotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values. [ABSTRACT FROM AUTHOR]

Using microalgae as a feedstock for biofuel is a method to reduce carbon dioxide emissions. Nannochloropsis is among the most promising microalgal taxa for biofuel production. To improve the lipid productivity of the marine microalga Nannochloropsis sp. SCSIO-45217 a novel method involving phosphate (NaH2PO4·2H2O) adjustment was established. First, the effects of phosphorous concentration on the lipid accumulation and biochemical composition of Nannochloropsis sp. SCSIO-45217 was studied in a batch culture. Subsequently, to enhance lipid productivity, a fed-batch cultivation mode was established with an initial phosphate concentration of 8 mg L−1 and continuous supplementation with 1, 2 and 3 mg L−1 phosphate, respectively, beginning on the 4th day. In batch culture the lipid content increased gradually from 27.45 to 46.15% DW with decreasing phosphate concentrations. Along with the decrease in phosphate concentrations, the contents of neutral lipids, eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) increased from 50.87 to 70.07%, 2.06 to 7.30% and 2.91 to 11.73% of the total lipid content, respectively. Lipid productivity was enhanced up to 127.20 mg L−1 day−1 in fed-batch culture, which was approximately threefold-fold higher than that of the low phosphate group (8 mg L−1). The productivity was 43.2% higher than that of the high phosphate group (40 mg L−1). These results indicated that Nannochloropsis sp. SCSIO-45217 can be a promising source for PUFAs and the optimized supply of phosphate in fed-batch cultivation is a suitable cultivation strategy for microalgal lipid production. [ABSTRACT FROM AUTHOR]

Nannochloropsis, due to its high lipid content, small size and growth rate, can be exploited for the production of biofuel and other value-added products. This study analyzed synergistic responses of high irradiance and nitrogen deprivation on Nannochloropsis cells to enhance biomass and lipid production. The growth of Nannochloropsis was first optimized under different irradiance 50, 100, 150, 200 and 250 μmol photons m-2 s-1. The maximum specific growth rate (0.25 day-1) and dry weight (0.67 g L-1) were obtained (p<0.05) at photosynthetically active radiation (PAR) of 200 µmol photons m-2 s-1, further increase in light intensity decreased the growth rate and dry weight up to 0.2 day-1 and 0.41g L-1, respectively. The stress of high light for a short period increased the lipid content by 51.1% and decreased the protein content by 35.2% over the control values. In the second phase of the study, the combined stress of high light intensity of 200 µmol photons m-2 s-1 PAR and nitrate depletion (50%, 75%, 100% N-) were studied and observed significant (p<0.05) increase in lipid content from 21.3±1.4% to 43.2±1.56% DCW and decrease in protein from 30.7±1.3% to 16.3±1.2% DCW, respectively. The highest lipid productivity (140 mg L-1 d-1) had been observed in a culture grown at 75% N- starved cells (p<0.05). However, the further increase of nitrate deprivation to 100% decreased the lipid productivity to 107 mg L-1 d-1. Maximum accumulations of TG (19.6% DCW) were observed on 6 days (early stationary phase) in a 100% nitrogen deprived medium, although significant accumulation was observed on 4 days and 6 days of nitrate reduction. [ABSTRACT FROM AUTHOR]

The genus Nannochloropsis (Eustigmatophyceae), including N. oceanica, has attracted considerable interest, due to its ability to accumulate high levels of lipids. Under large-scale cultivation outdoors, cells are exposed to a combination of high oxygen and light irradiance conditions that may promote photoinhibition, so selecting strains able to tolerate oxidative stress is desirable. Rose bengal (RB) reacts with oxygen to form singlet oxygen in the presence of light. Therefore, RB can be a very useful tool for testing the sensitivity of algae to oxidative stress and thus identify strains capable of photosynthesis under oxidative stress. In this study, two mutants of N. oceanica, RB2 and RB113, which are resistant to high concentrations of rose bengal, were phenotypically characterized for their sensitivity to high oxygen concentrations. Both strains exhibited levels of catalase and ascorbate peroxidase 1.5- and 3-fold higher, respectively, than the wild type. In addition, the synthesis of carotenoids was lower than in the wild type, indicating lower oxidative stress. These results were confirmed by chlorophyll fluorescence measurements, which indicated a lower sensitivity of the photosynthetic apparatus of the mutants, especially of RB2, even in the presence of H2O2. The results support the hypothesis that resistance to rose bengal may also induce resistance to high dissolved oxygen stress. The ability shown by the mutants to perform photosynthesis more efficiently under high oxygen stress than the wild type makes them promising candidates for outdoor cultures. [ABSTRACT FROM AUTHOR]

The overall goal of this study was to provide solutions to innovative microalgae-based technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second “stress” phase using the RAS wastewater to enhance the production of high-value metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and β-carotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values.

In light of the escalating global energy crisis, microalgae have emerged as highly promising producers of biofuel and high-value products. Among these microalgae, Nannochloropsis has received significant attention due to its capacity to generate not only triacylglycerol (TAG) but also eicosapentaenoic acid (EPA) and valuable carotenoids. Recent advancements in genetic tools and the field of synthetic biology have revolutionized Nannochloropsis into a powerful biofactory. This comprehensive review provides an initial overview of the current state of cultivation and utilization of the Nannochloropsis genus. Subsequently, our review examines the metabolic pathways governing lipids and carotenoids, emphasizing strategies to enhance oil production and optimize carbon flux redirection toward target products. Additionally, we summarize the utilization of advanced genetic manipulation techniques in Nannochloropsis. Together, the insights presented in this review highlight the immense potential of Nannochloropsis as a valuable model for biofuels and synthetic biology. By effectively integrating genetic tools and metabolic engineering, the realization of this potential becomes increasingly feasible.

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

The growing application of nanotechnology has led to the release of nanoparticles in aquatic ecosystems. Thus, investigating the effect of nanoparticles on microalgae, as the primary producers of the food chain, is an important issue. The present study was conducted to examine the toxicity mechanisms of α- Fe2O3 nanoparticles (Fe2O3-NPs) dominated by oxidative stress in Nannochloropsis oculata. The accumulation of Fe2O3-NPs on the cell surface of N. oculata and in f2 medium was observed in SEM images and DLS analysis, respectively. EDX graph and TEM images also showed the presence of NPs in N. oculata. There was an increase of H2O2 and MDA content and growth inhibition was also elevated in N. oculata exposed to Fe2O3-NPs. Further, Fe2O3-NPs application caused a noticeable enhancement of non-enzymatic antioxidants (carotenoids, phenolic compounds and PUFAs) and APX activity, while the content of SFAs and MUFAs as well as the activity of CAT and PPO were reduced as a result of Fe2O3-NPs stress. Overall, the activation of defence mechanisms was not effective in N. oculata and treatment with concentrations of more than 50 mg l−1 Fe2O3-NPs could induce oxidative stress in this microalga. [ABSTRACT FROM AUTHOR]

The photosynthetic apparatus is a major reactive oxygen species (ROS) proliferator, especially in high-light environments. The role of ROS in photoinhibition and photoacclimation mechanisms has been extensively explored, primarily in model plant species. However, little work has been performed on the topic in non-Archaeplastida organisms, such as the model heterokont species Nannochloropsis oceanica. To investigate the photoacclimation and damaging impact of singlet oxygen and superoxide anions on the photosynthetic apparatus of N. oceanica, we subjected cells to two doses of methyl viologen and rose bengal. Significant findings: Rose bengal (a singlet-oxygen photosensitizer) induced changes to the photosynthetic apparatus and PSII photochemistry mirroring high-light-acclimated cells, suggesting that singlet-oxygen signaling plays a role in the high-light acclimation of PSII. We further suggest that this singlet-oxygen pathway is mediated outside the plastid, given that rose bengal caused no detectable damage to the photosynthetic apparatus. Methyl viologen (a superoxide-anion sensitizer) induced an enhanced non-photochemical quenching response, similar to what occurs in high-light-acclimated cells. We propose that superoxide anions produced inside the plastid help regulate the high-light acclimation of photoprotective pathways. [ABSTRACT FROM AUTHOR]

The cell cycle is the fundamental cellular process of eukaryotes. Although cell-cycle-related genes have been identified in microalgae, their cell cycle progression differs from species to species. Cell enlargement in microalgae is an essential biological trait. At the same time, there are various causes of cell enlargement, such as environmental factors, especially gene mutations. In this study, we first determined the phenotypic and biochemical characteristics of a previously obtained enlarged-cell-size mutant of Nannochloropsis oceanica, which was designated ECS. Whole-genome sequencing analysis of the insertion sites of ECS indicated that the insertion fragment is integrated inside the 5′-UTR of U/P-type cyclin CYCU;1 and significantly decreases the gene expression of this cyclin. In addition, the transcriptome showed that CYCU;1 is a highly expressed cyclin. Furthermore, cell cycle analysis and RT-qPCR of cell-cycle-related genes showed that ECS maintains a high proportion of 4C cells and a low proportion of 1C cells, and the expression level of CYCU;1 in wild-type (WT) cells is significantly increased at the end of the light phase and the beginning of the dark phase. This means that CYCU;1 is involved in cell division in the dark phase. Our results explain the reason for the larger ECS size. Mutation of CYCU;1 leads to the failure of ECS to fully complete cell division in the dark phase, resulting in an enlargement of the cell size and a decrease in cell density, which is helpful to understand the function of CYCU;1 in the Nannochloropsis cell cycle.

Low/high temperature stress is one of the key abiotic factors restricting the implementation of microalgal large-scale production. Metabolism, involved in abiotic stress resistances, have been reported in plants, but the related research in microalgae is far behind. This study analyzed the transcriptional changes in Nannochloropsis oceanica under low and high temperature stresses by RNA-seq technology. Results indicated that the total differentially expressed gene (DEG) amount under high temperature stress was much more than low temperature stress. GO enrichment analysis suggested that DEGs under low temperature were mainly concentrated in protein modification and phosphorus metabolic process, while transmembrane transport, lipid localization and transport, and cellular homeostasis were the most enriched terms under high temperature. Moreover, “membrane” was the most enriched term in both low and high temperature groups. The gene expressions of 37 stress resistance-related enzymes/proteins (172 genes) were analyzed concretely in N. oceanica for the first time. Results revealed that ornithine decarboxylase (ODC) was significantly down-regulated, while choline dehydrogenase (CHDH), late embryogenesis abundant (LEA), dehydrin (DHN), chitinase (CHI), calmodulin (CaM), and lipid transfer protein (LTP) were extremely up-regulated under low temperature. Under high temperature, more genes with drastic changes were identified. Superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione synthetase (GSS), and glyoxalase II (Gly II) exhibited down-regulated significantly, while glutathione S-transferase (GST), CHDH, L-asparaginase (L-ASP), LEA, DHN, early response to dehydration protein (ERD), heat shock protein (HSP), and CaM were dramatically up-regulated. No significant changes were detected in glutathione peroxidase (GPX), tocopherol cyclase (TC), gamma-glutamylcysteine synthetase (γ-GCS), pyrroline‐5–carboxylate reductase (P5CR), asparagine synthetase (ASN ), succinate-semialdehyde dehydrogenase (SSADH), pyruvate decarboxylase (PDC), and S-adenosylmethionine synthetase (SAMS) under low or high temperature. Furthermore, 166 transcription factor (TF) genes related to plant stress resistance were characterized and categorized into 20 TF families. MYB was the most abundant and active one, revealing that the MYB family played a critical role of temperature resistance in N. oceanica. These new findings could benefit the understanding of the temperature resistance mechanism in Nannochloropsis. The significantly changed genes might be ideal candidates for breeding elite Nannochloropsis strains with stronger resistance to temperature stress using genetic engineering methods.

Oleaginous microalgae represent potential feedstocks for the sustainable production of lipids thanks to their ability to accumulate triacylglycerols (TAGs). TAG accumulation in several algal species is strongly induced under specific conditions such as nutrient deprivation and high light which, however, also negatively impact growth. Genetic modification of lipogenic pathways can potentially enhance TAG accumulation without negatively affecting growth, avoiding the trade-off between biomass and lipid productivity. In this study, the phospholipid: diacylglycerol acyltransferase (PDAT), an enzyme involved in membrane lipid recycling, was overexpressed in the seawater alga Nannochloropsis gaditana. PDAT overexpression induced increased TAG content in actively growing algae cultures while no effects were observed in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation. The increase of TAG content was confirmed also in a strain cultivated in industrially relevant conditions even though PDAT overexpression, if too strong, the gene overexpression becomes detrimental for growth in the longer term. Results overall suggest that genetic modulation of the PDAT gene represents a promising strategy to increase microalgae lipid content by minimizing negative effects on biomass productivity. • PDAT was overexpressed in Nannochloropsis gaditana. • Overexpression induced accumulating of triacylglycerols. • Increased lipids accumulation was confirmed in long term photobioreactors cultures. • Strong overexpression showed negative impact on growth, only visible on long term. [ABSTRACT FROM AUTHOR]

• We propose a predictor (termed DNPPro) based on densely connected convolutional neural networks to identify the promoter of Nannochloropsis. • We construct negative samples in a way called within-group scrambling to overcome the limitations of randomly selecting a non-promoter region from the same genome as the negative sample and improve the robustness of the method. • Benchmarking experiments shows that DNPPro is more efficient and can outperform current sequence processing methods. DNPPro can extract sequence features to a greater extent and be better applied to long sequences. • Visual analysis using T-SNE enhanced the interpretability of DNPPro. Promoter is a key DNA element located near the transcription start site, which regulates gene transcription by binding RNA polymerase. Thus, the identification of promoters is an important research field in synthetic biology. Nannochloropsis is an important unicellular industrial oleaginous microalgae, and at present, some studies have identified some promoters with specific functions by biological methods in Nannochloropsis, whereas few studies used computational methods. Here, we propose a method called DNPPro (D ense N et- P redict- Pro moter) based on densely connected convolutional neural networks to predict the promoter of Nannochloropsis. First, we collected promoter sequences from six Nannochloropsis strains and removed 80% similarity using CD-HIT for each strain to yield a reliable set of positive datasets. Then, in order to construct a robust classifier, within-group scrambling method was used to generate negative dataset which overcomes the limitation of randomly selecting a non-promoter region from the same genome as a negative sample. Finally, we constructed a densely connected convolutional neural network, with the sequence one-hot encoding as the input. Compared with commonly used sequence processing methods, DNPPro can extract long sequence features to a greater extent. The cross-strain experiment on independent dataset verifies the generalization of our method. At the same time, T-SNE visualization analysis shows that our method can effectively distinguish promoters from non-promoters. [ABSTRACT FROM AUTHOR]

A local strain of Nannochloropsis granulata (Ng) has been reported as the most productive microalgal strain in terms of both biomass yield and lipid content when cultivated in photobioreactors that simulate the light and temperature conditions during the summer on the west coast of Sweden. To further increase the biomass and the biotechnological potential of this strain in these conditions, mixotrophic growth (i.e., the simultaneous use of photosynthesis and respiration) with glycerol as an external carbon source was investigated in this study and compared with phototrophic growth that made use of air enriched with 1–2% CO2. The addition of either glycerol or CO2-enriched air stimulated the growth of Ng and theproduction of high-value long-chain polyunsaturated fatty acids (EPA) as well as the carotenoid canthaxanthin. Bioassays in human prostate cell lines indicated the highest antitumoral activity for Ng extracts and fractions from mixotrophic conditions. Metabolomics detected betaine lipids specifically in the bioactive fractions, suggesting their involvement in the observed antitumoral effect. Genes related to autophagy were found to be upregulated by the most bioactive fraction, suggesting a possible therapeutic target against prostate cancer progression. Taken together, our results suggest that the local Ng strain can be cultivated mixotrophically in summer conditions on the west coast of Sweden for the production of high-value biomass containing antiproliferative compounds, carotenoids, and EPA. [ABSTRACT FROM AUTHOR]

Microalgae have been highlighted as one of the promising feedstocks for renewable fuel production owing to their high growth rate, ability to grow on wastewater, and to convert CO2 into lipids, the main source of biodiesel. However, lipid extraction from microalgal cells is cumbersome due to its thick cell wall which precludes commercial biodiesel production from microalgae, and to improve the lipid extraction efficiency, optimization of the operational parameters is crucial. One of the well-known challenges is to develop an economical technique to obtain a high lipid extraction efficiency. Hence, this study aimed to investigate the optimized conditions of lipid extraction from marine species of Nannochloropsis sp. PTCC 6016 isolated from the Persian Gulf using Soxhlet, Bligh & Dyer, and ultrasonication methods. Initially, Nannochloropsis sp. was cultivated in a 1000L open pond for the purpose of lipid evaluation and process optimization, to be transferred to a 2000 L and subsequently, a 30000L open pond. Effects of various solvent ratios, mixing time and mixing frequency, pre-treatment, and cell age have been evaluated, and the optimal conditions have been determined. Lipid extraction prediction has been investigated by artificial neural network (ANN) and support vector regression (SVR) methods. Experimental results show that the maximum extraction yield of 0.56 gr extracted lipid/gr dry biomass was obtained using Soxhlet method after 90 days of cultivation using chloroform–methanol (1:1) as extraction solvents. However, considering 10 times higher solvent used in Soxhlet method compared to Bligh and Dyer and ultrasonication methods, the ultrasonication method was the most desired option for lipid extraction efficiency of Nannochloropsis sp. in large-scale operations. In this regard, cost-effective biofuel production should be considered as the focus of future studies to achieve sustainability, energy security and addressing the climate change due to high greenhouse gas emissions. [ABSTRACT FROM AUTHOR]

Nitrogen starvation is a high-efficiency strategy for inducing microalgae to accumulate lipids, polysaccharides, and carotenoids. Traditional nitrogen starvation that transfers microalgal cells into nitrogen depletion abruptly (AND) is labor-consuming and irreversibly damages microalgae. This study used gradual nitrogen depletion in medium (GND) to effectively induce carbohydrates, TAGs, and carotenoids accumulation in Nannochloropsis. After GND for 7 days, the carbohydrate and lipid contents were 1.34- and 0.15-fold higher than the initial content. In addition, astaxnathin and canthaxanthin contents were induced from 0 to 0.32 ± 0.03 mg/g DW and 0.09 ± 0.01 mg/g DW, respectively. Three days of GND revealed 158 significant differentially regulated proteins, including 113 up- and 45 down-regulated proteins. The proteins were involved in pathways for photosynthesis, carbon metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, and fatty acid biosynthesis. Gradual nitrogen depletion stimulated carbohydrate and TAG biosynthesis, fluxed more photosynthetic carbon into the carbohydrate reservoir and strengthened the TCA cycle and glyoxylate cycle. Transcriptional studies also showed that GND up-regulated the genes involved in carbohydrates and TAG biosynthesis. This study proposed a possible GND regulatory model for carbon repartition in Nannochloropsis. • GND effectively induces carbohydrates and TAG accumulation in N. oceanica. • GND stimulates carotenoid biosynthesis. • GND fluxes more photosynthetic carbon into the carbohydrate reservoir. • GND strengthens the TCA and glyoxylate cycles. • This study proposes a GND-regulatory model for N. oceanica. [ABSTRACT FROM AUTHOR]

Microalgae, recognized as sustainable and eco-friendly photosynthetic microorganisms, play a pivotal role in converting CO 2 into value-added products. Among these, Nannochloropsis salina (Microchloropsis salina) stands out, particularly for its ability to produce eicosapentaenoic acid (EPA), a crucial omega-3 fatty acid with significant health benefits such as anti-inflammatory properties and cardiovascular health promotion. This study focused on optimizing the cultivation conditions of Nannochloropsis salina to maximize EPA production. We thoroughly investigated the effects of varying temperatures and nitrogen (NaNO 3) concentrations on biomass, total lipid content, and EPA proportions. We successfully identified optimal conditions at an initial NaNO 3 concentration of 1.28 g.L−1 and a temperature of 21 °C. This condition was further validated by response surface methodology, which resulted in the highest EPA productivity reported in batch systems (14.4 mg.L−1.day−1). Quantitative real-time PCR and transcriptomic analysis also demonstrated a positive correlation between specific gene expressions and enhanced EPA production. Through a comprehensive lipid analysis and photosynthetic pigment analysis, we deduced that the production of EPA in Nannochloropsis salina seemed to be produced by the remodeling of chloroplast membrane lipids. These findings provide crucial insights into how temperature and nutrient availability influence fatty acid composition in N. salina , offering valuable guidance for developing strategies to improve EPA production in various microalgae species. [Display omitted] • NaNO 3 and temperature conditions were optimized for EPA production in N. salina. • Lipid and pigment analysis indicate EPA remodeling in chloroplast membranes. • A positive correlation was found between gene expression and EPA production. • Response surface methodology confirms ideal conditions for EPA synthesis. [ABSTRACT FROM AUTHOR]

Improved methods for the extraction of eicosapentaenoic acid (EPA), an essential and economically important polyunsaturated fatty acid, are urgently required. However, lipid extraction rates using food-grade solvents such as ethanol are usually low. To improve the ethanol-based extraction rate, and to elucidate the relevant mechanisms, we used cellulase and laccase to treat powdered Nannochloropsis, one of the most promising microalgal sources of EPA. Cellulase and laccase synergistically increased lipid yields by 69.31% and lipid EPA content by 42.63%, by degrading the amorphous hemicellulose and cellulose, improving crystallinity, and promoting the release and extraction of lysodiacylglyceryltrimethylhomoserine. Scanning electron microscopy showed that cell morphology was substantially altered, with cell-wall rupture, loss of cell boundaries, and the release of intracellular substances. In conclusion, Nannochloropsis lipid yields may be directly linked to cell-wall hemicellulose structure, and enzymatic treatment to alter this may improve lipid yields. [ABSTRACT FROM AUTHOR]

In the present study, the effect of three light wavelengths including blue, red, and white light and three light intensities (1000, 2000, 3000 LUX) were investigated on the growth rate, duplication time, and biomass production in microalgae Nannochloropsis oculata. Different treatments of algal cultures were cultured under 25 ppt salinity at temperature 24 ºC, and photoperiod cycle 12:12 (light:dark) using Guillard medium. The whole period of this experiment was 12 days. Cell count was conducted every other day and the biomass was measured on the last day in different treatments. The highest cell concentration (33×106 cell ml-1) was obtained in treatment with blue light and 2000 LUX after 10 days of culture. The maximum specific growth rate (0.40±0.01 d-1) was observed at the blue light and 3000 LUX and also the minimum duplication time occurred at this treatment (1.16±0.03 d-1). The maximum biomass (0.54± 0.003 gl-1) was recorded at the blue light and 2000 LUX. According to the results, the best light condition for adaptation of microalga N. oculata to cultural systems and obtaining maximum cell concentration and biomass is blue light with 2000-3000 LUX light intensity. Therefore, this light regime is recommended for N. oculata culture in phycolabs and aquaculture systems.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non-photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor-intensive and time-consuming generation of stable transgenic lines and mutant populations - approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ-related genes from diverse organisms, we performed Agrobacterium tumefaciens-mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.

Microalgae are used in food and feed, and they are considered a potential feedstock for sustainably produced chemicals and biofuel. However, production of microalgal-derived chemicals is not yet economically feasible. Genetic engineering could bridge the gap to industrial application and facilitate the production of novel products from microalgae. Here, we report the discovery of a novel gene expression system in the oleaginous microalga Nannochloropsis that exploits the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation. We identified the nucleolus as a genomic safe harbor for Pol I transcription and used it to construct transformant strains with consistently strong transgene expression. The new expression system provides an outstanding tool for genetic and metabolic engineering of microalgae and thus will probably make substantial contributions to microalgal research. This work describes the development of a novel system for genetic engineering of a eukaryotic microalga. It was shown that eukaryotic RNA polymerase I can be exploited for expression of protein-coding genes by employing an internal ribosome entry site to allow translation. Importantly, this study identified the nucleolus as a genomic safe harbor for consistent and strong gene expression. [ABSTRACT FROM AUTHOR]

Fatty acid profile was one of the important characteristic parameters of edible oil. With the intensification of interest in microalgae as a potential source of edible oil, rapid methods are needed to determine the fatty acid composition of microalgae. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was applied to determine the lipid and fatty acid contents of Nannochloropsis oceanica, a species rich in eicosapentaenoic acid. Samples with different intracellular components were obtained by setting different culture conditions (light intensity and nitrogen concentration). In the spectra of FTIR, some characteristic bands for lipids (3025–2800 cm−1, 1770–1710 cm−1, 1480–1350 cm−1) were identified. Partial least square regression (PLSR) models were developed to predict total biomass, lipid contents, and main fatty acid contents (palmitic acid C16:0, palmitoleic acid C16:1, eicosatetraenoic acid C20:4, eicosapentaenoic acid C20:5). Generally, the prediction ability of the model established with full spectra was slightly better than that of the model established with specific bands. In the optimized model, excellent coefficients of determination (R2 ≥ 0.90), both for calibration and prediction, were found for all variables, except for fatty acid C20:4, which was slightly worse (R2 = 0.8797). The pretreatment methods of samples were further studied. It was found that the acid-heating method (0.1 M HCl, 100 °C, 0.5 h) effectively reduced the interference of the carbohydrate in samples, thus improving the measurement of the characteristic fatty acids C20:4 and C20:5 of N. oceanica. This study showed the feasibility of FTIR as a rapid screening method for evaluating biomass and lipid content, especially some special fatty acid profile of microalgae. [ABSTRACT FROM AUTHOR]

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGAT s had no or slight effect on EPA abundance in TAG, introducing selected DGAT s with strong activity on EPA-CoA, particularly the Chlamydomonas -derived CrDGTT1 , which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well. • Screening of over 30 algal DGATs revealed potent enzymes acting on EPA-CoA. • Overexpressing selected DGAT s boosted EPA abundance and level in TAG in algae. • Co-overexpressing CrDGTT1 and Δ0-ELO1 further enhanced TAG-derived EPA level. • EPA was enriched in algal TAG by use of combined pulling and pushing strategies. [ABSTRACT FROM AUTHOR]

A feeding experiment was conducted to survey proper microalgae species for nonfish meal and non-fish oil diet of red sea bream. Five iso-nitrogenous and iso-lipidic diets were formulated (CP 47%, CL 19%). Fish oil formulated non-fish meal diet was arranged as a control diet with soybean meal, soy protein concentrate and corn gluten meal (FO). Fish oil of FO was replaced by Nannochloropsis meal and Schizochytrium meal for each of two diets (NAN, SCH). Both microalgae meals were mixed in a diet (NS), and Chlorella meal was formulated in NS diet to replace plant protein source (NSC). Duplicated groups of juvenile red sea bream (5.9 g) were fed the experimental diets for 75 days to near satiation. The growth was the highest in NS and NSC diet group. The other algae meal formulated diets fed groups showed the similar growth performance to FO. In fatty acid profiles, NAN group showed the lowest n-3 LC-PUFA with low DHA content in fatty acid composition of total lipid in fish carcass and hepatic PL, but the other algae meal diet group showed no significant difference with FO group. The results might suggest blending microalgae and using them with high amount is a key strategy for development of non-fish meal and non-fish oil diet for red sea bream. [ABSTRACT FROM AUTHOR]

El estudio se centró en contribuir a la Bioeconomía Acuícola evaluando el ritmo reproductivo de Nannochloropsis oculata en dos baterías experimentales con concentraciones salinas de 33 ‰, 25 ‰, 20 ‰, 15 ‰, 10 ‰ y 5 ‰. A cada una se le aplicó dosis únicas de 0.32 ‰ y 1 ‰ de F/2 Guillard, respectivamente. Se realizaron dos experimentos en tiempos de 10 y 23 días a temperatura de 25 ºC. El primer experimento con 0.32 ‰ de F/2 Guillard, N. oculata presentó mayor número de cel/ml en salinidades de 33 ‰ y 25 ‰, a los 8 días de estudio, denotando la afinidad de N. oculata de reproducirse con mayor velocidad en ese rango de salinidad. La importancia del uso de F/2 Guillard fue observada usando 1‰ debido a que la microalga presenta similar número de cel/ml (P≤0.05) en concentraciones salinas de 33 ‰, 25 ‰, 20 ‰ y 15 ‰ y similar concentración a las microalgas cultivadas con 0.32 ‰ de F/2 Guillard en salinidades de 33 ‰ y 25 ‰. La capacidad de N. oculata fue testado en un segundo experimento donde los resultados muestran similar número de cel/ml entre los tratamientos salinos, en ambas baterías, a los 23 días de cultivo. Los resultados muestran que N. oculata puede ser cultivada con bajas concentraciones de F/2 Guillard en rangos de salinidad entre 25 ‰ y 33 ‰, implica reducción de costos.

Abstract Background Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. Results Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. Conclusions The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application.

Lysine lactylation (Kla) is a kind of novel post-translational modification (PTM) that participates in gene expression and various metabolic processes. Nannochloropsis has a remarkable capacity for triacylglycerol (TAG) production under nitrogen stress. To elucidate the involvement of lactylation in lipid synthesis, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) and mRNA-seq analyses to monitor lactylation modifications and transcriptome alterations in Nannochloropsis oceanica . In all, 2057 genes showed considerable variation between nitrogen deprivation (ND) and nitrogen repletion (NR) conditions. Moreover, a total of 5375 differential Kla peaks were identified, including 5331 gain peaks and 44 loss peaks under ND vs NR. The differential Kla peaks were primarily distributed in the promoter (≤1 kb) (71.07%), 5'UTR (22.64%), and exon (4.25%). Integrative analysis of ChIP-seq, transcriptome, and previous proteome and lactylome data elucidates the potential mechanism by which lactylation promotes lipid accumulation under ND. Lactylation facilitates autophagy and protein degradation, leading to the recycling of carbon into the tricarboxylic acid (TCA) cycle, thereby providing carbon precursors for lipid synthesis. Additionally, lactylation induces the redirection of carbon from membrane lipids to TAG by upregulating lipases and enhancing the TCA cycle and β-oxidation pathways. This research offers a new perspective for the investigation of lipid biosynthesis in Nannochloropsis .

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis , have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis , very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis , long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria , which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans -splicing in Monodopsis and Vischeria at 36–61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae. [ABSTRACT FROM AUTHOR]