Search

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]

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]

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

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]

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 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 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]

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]

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]

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.

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]

Microalgae are known producers of antioxidant and anti-inflammatory compounds, making them natural alternatives to be used as food and feed functional ingredients. This study aimed to valorise biomass and exploit new applications and commercial value for four commercially available microalgae: Isochrysis galbana, Nannochloropsis sp., Tetraselmis sp., and Phaeodactylum tricornutum. For that, five extracts were obtained: acetone (A), ethanol (E), water (W), ethanol:water (EW). The antioxidant capacity (ABTS•+/DPPH•/•NO/O2•−/ORAC-FL) and anti-inflammatory capacity (HBRC/COX-2) of the extracts were screened. The general biochemical composition (carbohydrates, soluble proteins, and lipids) and the main groups of bioactive compounds (carotenoids, phenolic compounds, and peptides) of extracts were quantified. The results of antioxidant assays revealed the potential of some microalgae extracts: in ABTS•+, Nannochloropsis sp. E and Tetraselmis sp. A, E, and P; in DPPH•, Tetraselmis sp. A and E; in •NO, P. tricornutum E and EW; in O2•−, Tetraselmis sp. W; and in ORAC-FL, I. galbana EW and P. tricornutum EW. Concerning anti-inflammatory capacity, P. tricornutum EW and Tetraselmis sp. W showed a promising HBRC protective effect and COX-2 inhibition. Hence, Tetraselmis sp. and P. tricornutum extracts seem to have potential to be incorporated as feed and food functional ingredients and preservatives.

In this study, the effect of different concentrations of CuO nanoparticles (0, 5, 10, 50, 100, 200 mg/L) was investigated on Nannochloropsis oculata. The results showed that the viability percentage and Chlorophyll a content was significantly decreased in the algal cells treated with CuO nanoparticles (CuO-NPs) compared to the control. The content of protein remarkable was decreased in response to CuO-NPs, while the level of phenolic compounds significantly was increased in comparison to the control. The activity of polyphenol oxidase (PPO) enzyme was raised in N. oculata cells with the increase of CuO-NPs concentration, which was accordance with the results of phenolic compounds. Also, the algal cell aggregation and the cell membrane damage were observed in the images of scanning electron microscopy (SEM). Increase of phenolic compounds and PPO activity as well as the cell aggregation probably were defense methods in this microalga in response to the stress from NPs.

Abstract Background Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR–Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout. Results In this study, we report for the first time an alternative approach for CRISPR–Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines. Conclusion We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Today’s global problems and challenges have given rise to a new field of interest – bioeconomics. It is strongly related to phycoprospecting, or searching for specific algal strains of commercial importance. There has been growing interest in the small algal class Eustigmatophyceae in recent years. These microscopic stramenopilic algae, which have all the advantages of microalgal cultivation, have proved to be promising commercial sources of valuable compounds (e.g. carotenoids, unsaturated fatty acids, amino acids) in aquaculture, agriculture, biofuels production, medicine, pharmaceutics, cosmetics, wastewater treatment, environmental control, etc. The present review shows the main genera and strains of commercial importance, outlines their main fields of application and some gaps in our knowledge in this aspect. Today, the great promising bioeconomical potential of these algae has generally been recognized, but in the present state of its infancy, it is far from being fully exploited.

This study was designed to evaluate the effect of Nannochloropsis oculata microalgae supplementation on 120 rainbow trout fingerling (initial weight of 2±0.3 g). Fish were randomly distributed into four groups and stocked into 12 aquaria (20 liter) in triplicates. Four experimental diets contained 0 (control), 7.5 (T1), 15 (T2), and 22.5 (T3) g of the algae powder per kg of feed for 3 months. Lipid, liver enzymes, and some hematological properties of blood (i.e. WBC, RBC, HCT, MCV, HB, and MCHC) were measured at the end of the trial. Results showed that leucocyte population was not significantly different in all treatments (p>0.05). However, the highest erythrocyte counts and hematocrit index were obtained in T2 (p

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.

The Heterokonta or Stramenopile phylum comprises clades of unicellular photosynthetic species, which are promising for a broad range of biotechnological applications, based on their capacity to capture atmospheric CO2 via photosynthesis and produce biomolecules of interest. These molecules include triacylglycerol (TAG) loaded inside specific cytosolic bodies, called the lipid droplets (LDs). Understanding TAG production and LD biogenesis and function in photosynthetic stramenopiles is therefore essential, and is mostly based on the study of a few emerging models, such as the pennate diatom Phaeodactylum tricornutum and eustigmatophytes, such as Nannochloropsis and Microchloropsis species. The biogenesis of cytosolic LD usually occurs at the level of the endoplasmic reticulum. However, stramenopile cells contain a complex plastid deriving from a secondary endosymbiosis, limited by four membranes, the outermost one being connected to the endomembrane system. Recent cell imaging and proteomic studies suggest that at least some cytosolic LDs might be associated to the surface of the complex plastid, via still uncharacterized contact sites. The carbon length and number of double bonds of the acyl groups contained in the TAG molecules depend on their origin. De novo synthesis produces long-chain saturated or monounsaturated fatty acids (SFA, MUFA), whereas subsequent maturation processes lead to very long-chain polyunsaturated FA (VLC-PUFA). TAG composition in SFA, MUFA, and VLC-PUFA reflects therefore the metabolic context that gave rise to the formation of the LD, either via an early partitioning of carbon following FA de novo synthesis and/or a recycling of FA from membrane lipids, e.g., plastid galactolipids or endomembrane phosphor- or betaine lipids. In this review, we address the relationship between cytosolic LDs and the complex membrane compartmentalization within stramenopile cells, the metabolic routes leading to TAG accumulation, and the physiological conditions that trigger LD production, in response to various environmental factors.

The aim of the present work was focused on optimising the yield and quality of the biocrude obtained by hydrothermal liquefaction (HTL) of Nannochloropsis gaditana. Temperature, reaction time and microalga concentration were the variables used to carry out an experimental factorial design with a central composite design. The responses chosen were the biocrude yield and the nitrogen and oxygen content in the biocrude phase. A second-order model was obtained to predict the responses as a function of these variables. Temperature is the most determining factor with a positive influence on biocrude yield. The maximum biocrude yield (42.3 ± 0.8 wt%) was obtained at 320 °C, 10 min of reaction and 10 wt% microalgae concentration, and the nitrogen and oxygen content significantly decreased with respect to their corresponding levels in the initial microalgal biomass. The HHV value of the biocrude was 35.7 MJ/kg. The biocrude was composed of 30% of linear and branched hydrocarbons. [ABSTRACT FROM AUTHOR]

Temperature and light are two of the most crucial factors for microalgae production. Variations in these factors alter their growth kinetics, macromolecular composition and physiological properties, including cell membrane permeability and fluidity. The variations define the adaptation mechanisms adopted by the microalgae to withstand changes in these environmental factors. In the Qatar desert the temperature varies widely, typically between 10° and 45 °C There are also wide variations in light intensity, with values of over 1500 μmolhν.m−2s−1 in summer. A study of the effects of these thermal and light fluctuations is therefore essential for large-scale outdoor production systems, especially during the summer when temperature and light fluctuations are at their highest. The aim of this work is to study the impact of temperature and light intensity variations as encountered in summer period on the Nannochloropsis QU130 strain, which was selected for its suitability for outdoor cultivation in the harsh conditions of the Qatar desert. It was carried out using lab-scale photobioreactors enabling simulation of both constant and dynamic temperature and light regimes. Biomass productivity, cell morphology and biochemical compositions were examined first in constant conditions, then in typical outdoor cultivation conditions to elucidate the adjustments in cell function in respect of fluctuations. The dynamic light and temperature were shown to have interactive effects. The application of temperature cycles under constant light led to a 13.6% increase in biomass productivity, while a 45% decrease was observed under light and temperature regimes due to the combined stress. In all cases, the results proved that N. sp. QU130 has a high level of adaptation to the wide fluctuations in light and temperature stress. This was shown through its ability to easily change its physiology (cell size) and metabolic process in response to different cultivation conditions. [ABSTRACT FROM AUTHOR]

The Heterokonta or Stramenopile phylum comprises clades of unicellular photosynthetic species, which are promising for a broad range of biotechnological applications, based on their capacity to capture atmospheric CO2 via photosynthesis and produce biomolecules of interest. These molecules include triacylglycerol (TAG) loaded inside specific cytosolic bodies, called the lipid droplets (LDs). Understanding TAG production and LD biogenesis and function in photosynthetic stramenopiles is therefore essential, and is mostly based on the study of a few emerging models, such as the pennate diatom Phaeodactylum tricornutum and eustigmatophytes, such as Nannochloropsis and Microchloropsis species. The biogenesis of cytosolic LD usually occurs at the level of the endoplasmic reticulum. However, stramenopile cells contain a complex plastid deriving from a secondary endosymbiosis, limited by four membranes, the outermost one being connected to the endomembrane system. Recent cell imaging and proteomic studies suggest that at least some cytosolic LDs might be associated to the surface of the complex plastid, via still uncharacterized contact sites. The carbon length and number of double bonds of the acyl groups contained in the TAG molecules depend on their origin. De novo synthesis produces long-chain saturated or monounsaturated fatty acids (SFA, MUFA), whereas subsequent maturation processes lead to very long-chain polyunsaturated FA (VLC-PUFA). TAG composition in SFA, MUFA, and VLC-PUFA reflects therefore the metabolic context that gave rise to the formation of the LD, either via an early partitioning of carbon following FA de novo synthesis and/or a recycling of FA from membrane lipids, e.g., plastid galactolipids or endomembrane phosphor- or betaine lipids. In this review, we address the relationship between cytosolic LDs and the complex membrane compartmentalization within stramenopile cells, the metabolic routes leading to TAG accumulation, and the physiological conditions that trigger LD production, in response to various environmental factors. [ABSTRACT FROM AUTHOR]

Summary: Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community‐based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi‐omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single‐cell.cn). Via the Tripal toolkit, it features user‐friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae. Significance Statement: Nannochloropsis species have emerged as model organisms for microalgal systems and synthetic biology, but a centralized portal to store, mine and disseminate the rapidly expanding multi‐omics datasets is lacking. Here, we initiate an international consortium and present a comprehensive resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single‐cell.cn). NanDeSyn hosts genomic and epigenomic resources such as gene annotation, transcriptome, proteome, small RNA and mutant information, and provides tools for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis and a genome browser. [ABSTRACT FROM AUTHOR]

For efficient downstream processing, harvesting remains as one of the challenges in producing Nannochloropsis biomass, a microalga with high-value omega-3 oils. Flocculation is an effective, low-energy, low-cost method to harvest microalgae. Chitosan has been shown to be an effective food-grade flocculant; however, commercial chitosan is sourced from crustaceans, which has disadvantages including concerns over heavy-metal contamination. Thus, this study tests the flocculation potential of mushroom chitosan. Our results indicate a 13% yield of chitosan from mushroom. The identity of the prepared chitosan was confirmed by Fourier-transform infrared (FTIR) spectroscopy. Furthermore, results show that mushroom chitosan can be an alternative flocculant with >95% flocculation efficiency when tested in 100-mL jar and 200-L vertical column photobioreactor. Applications in a 2000-L raceway pond demonstrated that thorough mixing of mushroom chitosan with the algal culture is required to achieve efficient flocculation. With proper mixing, mushroom chitosan can be used to produce food-grade Nannochloropsis biomass suitable for the production of vegan omega-3 oils as a fish oil alternative.

Aquaculture is a fast-growing sector with a high scope for augmentation. Microalgae are frequently used for fish larvae culture. Hatcheries face many problems regarding the maintenance of a stock culture of the microalgae. Long term maintenance of microalgal species by serial sub culturing is often expensive, time-consuming and leads to genetic change. The present study is aimed to develop an alternative method for the long-term preservation of stock cultures. In this research, the cryopreservation protocol has been used for the preservation of Nannochloropsis sp at – 20° C using two criteria as different concentration of internal (Me2SO, MeOH, glycerol and ethylene glycol), external cryoprotectants (sucrose and sorbitol) and equilibration time (30 and 60 minutes). The results showed that the preservation of Nannochloropsis sp is feasible using this methodology. The present study proved that 10 % of Me2SO had the maximum viability at 30 minutes equilibration time and that the same concentration of cryoprotectant had resulted in the maximum viability after 6 months of preservation. From the study, it is evident that the microalgae can be preserved for a long time without using liquid nitrogen.

The richness of bioactive compounds in microalgae has long inspired their exploitation for human nutrition. However, very few species are authorized for this use to date. Microalgae of the genus Nannochloropsis are already exploited in aquaculture for their high content in PUFAs, carotenoids, polyphenols and vitamins. These molecules can contribute to delay aging processes and prevent the onset of many metabolic disorders triggered by unhealthy lifestyles and diets with an excess of saturated fatty acids. Moreover, Nannochloropsis is efficiently cultivated at an industrial scale in photobioreactors, without the use of pesticides and preventing biological or chemical contaminations. Here, information on its chemical composition, safety and toxicology is discussed. Nannochloropsis can be an eco-sustainable alternative to fishery products as source of bioactive compounds. Importantly, the daily consumption of few grams of biomass would result in a regular dietary intake of essential molecules for the prevention of cardiovascular pathologies and other disorders.

A dynamic coarse-grained model of microalgal growth considering light availability and temperature under discontinuous bioprocess operation was parameterized using experimental data from 15 batch cultivations of Nannochloropsis granulata in a pilot-scale tubular photobioreactor. The methodology applied consists of a consecutive two-step model parameter estimation using pooled, clustered and reorganized data to obtain initial estimates and multi-experiment fitting to obtain the final estimates, which are: maximum specific growth rate μmax = 1.56 d−1, specific photon half-saturation constant KS,ph = 1.89 molphgX-1d-1, specific photon maintenance coefficient mph = 0.346 molphgX-1d-1 and the cardinal temperatures Tmin = 2.3°C, Topt = 27.93°C and Tmax = 32.59°C. Biomass productivity prediction proved highly accurate, expressed by the mean absolute percent error MAPE = 7.2%. Model-based numerical optimization of biomass productivity for repeated discontinuous operation with respect to the process parameters cultivation cycle time, inoculation biomass concentration and temperature yielded productivity gains of up to 35%. This optimization points to best performance under continuous operation. The approach successfully applied here to small pilot-scale confirms an earlier one to lab-scale, indicating its transferability to larger scale tubular photobioreactors.

This study aims to determine the maximum specific biomass growth rates of Nannochloropsis gaditana and Tetraselmis chuii grown in sequential batch reactors. The maximum specific growth rates of N. gaditana and T. chuii were found to be 0.23 ± 0.04 gVSS/gVSS⋅d and 0.22 ± 0.05 gVSS/gVSS⋅d respectively. Optical density measured at 550 nm (OD550) accurately predict volatile suspended solids (VSS) concentration in N. gaditana growth media but not in T. chuii growth media. We observed decoupling of nitrate uptake from cell growth for both microalgae as growth continued well beyond the point where concentrations of nitrate in the growth media fell below the detection limit. This nitrate decoupling highlights the challenges in current microalgae growth models as growth was independent of the extracellular dissolved nitrogen in the system.

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 Background Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga, Nannochloropsis oceanica CCMP1779, and the oleaginous fungus, Mortierella elongata AG77, resulting in increased oil productivity. Results By growing separately and then combining the cells, the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the contents of polyunsaturated fatty acids (PUFAs) including arachidonic acid and docosahexaenoic acid in M. elongata. To increase TAG productivity in the alga, we developed an effective, reduced nitrogen-supply regime based on ammonium in environmental photobioreactors. Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, with ~ 15 and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering of N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bio-flocculation, this approach led to increased production of TAG under nutrient-replete conditions (~ 10% of DW) compared to the wild type (~ 6% of DW). Conclusions The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve biofuel productivity and allows for the engineering of polyunsaturated fatty acids.

Recently, there has been emerging interest in various natural products with skin protective effects as they are recognized as safe and efficient. Microalgae have developed chemical defense systems to protect themselves against oxidative stress caused by UV radiation by producing various bioactive compounds including a number of secondary metabolites, which have potential for cosmeceutical applications. In addition, microalgae have various advantages as a sustainable source for bioactive compounds with diverse functions due to their rapid growth rate, high productivity, and use of non-arable land. In this study, we aimed to investigate the cosmeceutical potential of ethanol extract from Nannochloropsis sp. G1-5 (NG15) isolated from the southern West Sea of the Republic of Korea. It contained PUFAs (including EPA), carotenoids (astaxanthin, canthaxanthin, β-carotene, zeaxanthin, violaxanthin), and phenolic compounds, which are known to have various skin protective functions. We confirmed that the NG15 extract showed various skin protective functions with low cytotoxicity, specifically anti-melanogenic, antioxidant, skin-moisturizing, anti-inflammatory, anti-wrinkling, and UV protective function, by measuring tyrosinase inhibition activity; melanin content; DPPH radical scavenging activity; expression of HAS-2, MMP-1, and Col1A1 genes; and elastase inhibition activity as well as cell viability after UV exposure. Our results indicated that the NG15 extract has the potential to be used for the development of natural cosmetics with a broad range of skin protective functions.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome‐editing system involving Platinum transcription activator‐like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon‐optimized for N. oceanica were synthesized, and their DNA‐binding activity was confirmed by single‐strand annealing assays in human HEK293T cells. All‐in‐one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome‐editing efficiency with no detectable off‐target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss‐of‐function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species. [ABSTRACT FROM AUTHOR]