Functional Characterization of Lycopene ß- and ε-Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60.

Lutein is a high-value carotenoid with many human health benefits. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently ß-cyclize both ends of lycopene to produce ß-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into ß-monocyclic γ-carotene and ultimately produced α-carotene with a ß-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

Unveiling the underlying molecular mechanisms of high lutein production efficiency in Chlorella sorokiniana FZU60 under a mixotrophy/photoautotrophy two-stage strategy by transcriptomic, physiological, and biochemical analyses.

BACKGROUND: Chlorella sorokiniana FZU60 is a promising lutein producing microalga. A mixotrophy/photoautotrophy two-stage strategy can achieve high biomass concentration at stage 1 and high lutein content at stage 2, leading to excellent lutein production efficiency in C. sorokiniana FZU60. However, the underlying molecular mechanisms are still unclear, restraining the further improvement of lutein production. RESULTS: In this study, physiological and biochemical analysis revealed that photochemical parameters (Fv/Fm and NPQ) and photosynthetic pigments contents increased during the shift from mixotrophy to photoautotrophy, indicating that photosynthesis and photoprotection enhanced. Furthermore, transcriptomic analysis revealed that the glyoxylate cycle and TCA cycle were suppressed after the shift to photoautotrophy, leading to a decreased cell growth rate. However, the gene expression levels of photosynthesis, CO2 fixation, autophagy, and lutein biosynthesis were upregulated at the photoautotrophy stage, demonstrating that microalgal cells could obtain more precursor to synthesize lutein for enhancing photosynthesis and reducing reactive oxygen species. CONCLUSIONS: The findings help to elucidate the molecular mechanisms for high lutein production efficiency of C. sorokiniana FZU60 under the mixotrophy/photoautotrophy strategy, identify key functional genes responsible for lutein biosynthesis, and shed light on further improvement of lutein production by genetic or metabolic engineering in future studies.

High-cell-density heterotrophic cultivation of microalga Chlorella sorokiniana FZU60 for achieving ultra-high lutein production efficiency.

Chlorella sorokiniana has received particular attention as a promising candidate for microalgal biomass and lutein production. In this work, heterotrophic cultivation was explored to improve the lutein production efficiency of a lutein-rich microalga C. sorokiniana FZU60. Flask cultivation results showed that the highest lutein productivity was achieved at 30°C with an initial cell concentration of 1.40 g/L. Furthermore, six types of fed-batch strategies based on nutrient composition and concentration were examined using a 5 L fermenter. Among them, ultra-high lutein production (415.93 mg/L) and productivity (82.50 mg/L/d) with lutein content of 2.57 mg/g were achieved with fed-batch 3F (i.e., pulse-feeding with concentrated urea-N medium to achieve a 3-fold nutrient concentration). The lutein production performance achieved is much higher than the reported values. This work demonstrates that heterotrophic cultivation of C. sorokiniana FZU60 with the proposed fed-batch strategy could significantly enhance the production performance and the commercial viability of microalgae-derived lutein.

Advancement of renewable energy technologies via artificial and microalgae photosynthesis.

There has been an urgent need to tackle global climate change and replace conventional fuels with alternatives from sustainable sources. This has led to the emergence of bioenergy sources like biofuels and biohydrogen extracted from microalgae biomass. Microalgae takes up carbon dioxide and absorbs sunlight, as part of its photosynthesis process, for growth and producing useful compounds for renewable energy. While, the developments in artificial photosynthesis to a chemical process that biomimics the natural photosynthesis process to fix CO2 in the air. However, the artificial photosynthesis technology is still being investigated for its implementation in large scale production. Microalgae photosynthesis can provide the same advantages as artificial photosynthesis, along with the prospect of having final microalgae products suitable for various application. There are significant potential to adapt either microalgae photosynthesis or artificial photosynthesis to reduce the CO2 in the climate and contribute to a cleaner and green cultivation method.

A Novel Algicidal Bacterium and Its Effects against the Toxic Dinoflagellate Karenia mikimotoi (Dinophyceae).

The toxic dinoflagellate Karenia mikimotoi is a harmful algal bloom-forming species in coastal areas around the world. It produces ichthyotoxins and hemolytic toxins, with deleterious effects on marine ecosystems. In this study, the bacterium Pseudoalteromonas sp. FDHY-MZ2, with high algicidal efficiency against K. mikimotoi, was isolated from a bloom event. Based on the results, it completely lysed K. mikimotoi cells within 24 h 0.5% (vol/vol), with the algicidal activity of the supernatant of the bacterium culture. Algal cell wall fragmentation occurred, leading to cell death. There was a marked decline in various photochemical traits. When treated with the supernatant, cellulase, pheophorbide a oxygenase (PAO) and cyclin B genes were significantly increased, suggesting induced cell wall deterioration, chloroplast degradation and cell cycle regulation of K. mikimotoi cells. In addition, the expression levels of reactive oxygen species (ROS) scavenging gene was significantly inhibited, indicating that the ROS removal system was damaged. The bacterial culture was dried to obtain the spray-dried powder, which showed algicidal activity rates of 92.2 and 100% against a laboratory K. mikimotoi culture and a field microcosm of Karlodinium sp. bloom within 24 h with the addition of 0.04% mass fraction powder. Our results demonstrate that FDHY-MZ2 is a suitable strain for K. mikimotoi and Karlodinium sp. blooms management. In addition, this study provides a new strategy for the anthropogenic control of harmful algal bloom-forming species in situ. IMPORTANCEK. mikimotoi is a noxious algal bloom-forming species that cause damaging of the aquaculture industry and great financial losses. Bacterium with algicidal activity is an ideal agency to inhibit the growth of harmful algae. In this approach application, the bacterium with high algicidal activity is required and the final management material is ideal for easy-to-use. The algicidal characteristics are also needed to understand the effects of the bacterium for managing strategy exploration. In this study, we isolated a novel algicidal bacterium with extremely high lysis efficiency for K. mikimotoi. The algicidal characteristics of the bacterium as well as the chemical and molecular response of K. mikimotoi with the strain challenge were examined. Finally, the algicidal powder was explored for application. The results demonstrate that FDHY-MZ2 is suitable for K. mikimotoi and Karlodinium sp. blooms controlling, and this study provides a new strategy for algicidal bacterium application.

Emerging biological wastewater treatment using microalgal-bacterial granules: A review.

Aiming at deepening the understanding of the formation and evolution of emerging microalgal-bacterial granule (MBG)-based wastewater treatment systems, the recent advances regarding the formation processes, transfer phenomena, innovative bioreactors development and wastewater treatment performance of MBG-based systems are comprehensively reviewed in this work. Particularly, the successful establishments of MBG-based systems with various inocula are summarized. Besides, as the indispensable factors for biochemical reactions in MBGs, the light and substrates (organic matters, inorganic nutrients, etc) need to undergo complicated and multi-scale transfer processes before being assimilated by microorganisms within MBGs. Therefore, the involved transfer phenomena and mechanisms in MBG-based bioreactors are critically discussed. Subsequently, some recent advances of MBG-based bioreactors, the application of MBG-based systems in treating various synthetic and real wastewater, and the future development directions are discussed. In short, this review helps in promoting the development of MBG-based systems by presenting current research status and future perspectives.

Role of nitrogen transport for efficient energy conversion potential in low carbon and high nitrogen/phosphorus wastewater by microalgal-bacterial system.

Microalgal-bacterial system (MBS) is potential biotechnology in wastewater treatment because it can remedy defects of conventional processes (e.g., insufficient carbon source and imbalanced elements ratio). However, the mechanisms of nitrogen (N) transport and removal in MBS are still unclear. In this study, it was discovered that MBS was conducive to adsorb NH4+-N and NO3--N through electrical neutralization, while extracellular polymeric substances (EPS) could provide binding sites (e.g., -OH and -CH3) for enhancing N transport and removal. The microalgae-bacteria interaction could accelerate N transport and removal from aqueous solution to cell. More importantly, the microalgal starch biosynthetic metabolism exhibited demonstrating the energy production potential could be boosted via MBS. Overall, the NO3--N and NH4+-N removal efficiencies, and energy yield were 82.28%, 94.15%, and 86.81 kJ/L, respectively, which are better than other relevant studies. Altogether, it is meaningful for revealing the applicability of MBS for treating wastewater and producing energy.

Insights into the microalgae-bacteria consortia treating swine wastewater: Symbiotic mechanism and resistance genes analysis.

This study investigated the effects of microalgae-bacteria consortia (MBC) (Chlorella pyrenoidosa-activated sludge (AS)) treating swine wastewater with low C/N ratios. After co-culture, the removal rates of NH4+-N and PO43--P increased by 53.84% and 43.52%. Furthermore, the sulfamethoxazole (SMX) degradation rates in MBC were slightly higher than in the activated sludge process. Interestingly, the absolute abundance of antibiotic resistance genes (ARGs) in effluent from MBC is relatively less than in the AS process. C. pyrenoidosa has a negative zeta potential that allows bacteria to adhere to its surface. The concentrations of carbohydrates and proteins in extracellular polymeric substance (EPS) of MBC dramatically increased compared with the AS process. At the phylum level, Proteobacteria, Bacteroidota, and Cyanobacteria were the main bacteria, while Ascomycota and Basidiomycota were the primary fungi in MBC. Overall, those findings lead to a better understanding of the swine wastewater containing antibiotic treatment by MBC.

Transcriptome responses of the dinoflagellate Karenia mikimotoi driven by nitrogen deficiency.

The availability of ambient N nutrient is often correlated with the occurrences of harmful algal bloom formed by certain dinoflagellates, making it important to understand how these species might be responding to such conditions. Here, transcriptome sequencing of Karenia mikimotoi was conducted to understand the underlying molecular mechanisms by which this dinoflagellate copes with nitrogen (N) deficiency. Transcriptomic analysis revealed 8802 unigenes (3.56%) that were differentially expressed with ≥ 2-fold change. Under N-depleted conditions, genes involved in glycolysis, fatty acid metabolism, and the tricarboxylic acid (TCA) cycle as well as lipid accumulation were significantly upregulated. The elevated expression of enzymes used in protein degradation and turnover suggests possible metabolic reconfiguration towards accelerated N recycling. Moreover, a significant increase in urea transporter was observed, indicating increased assimilation of organic nitrogen resources as an alternative in N-depleted cultures of K. mikimotoi. The down-regulated glutamate synthase genes were also identified under N deficiency, suggesting suppression of primary amino acid synthesis to save N resource. Taken together, results of this study show enhanced multiple N resource acquisition and reuse of multiple N resources constitute a comprehensive strategy to cope with N deficiency in a dinoflagellate.

Enhancement of co-production of lutein and protein in Chlorella sorokiniana FZU60 using different bioprocess operation strategies.

Co-production of multiple compounds is an efficient approach to enhance the economic feasibility of microalgae-based metabolites production. In this study, Chlorella sorokiniana FZU60 was cultivated under different bioprocess strategies to enhance the co-production of lutein and protein. Results showed that both lutein and protein content (7.72 and 538.06 mg/g, respectively) were highest at the onset of nitrogen deficiency under batch cultivation. Semi-batch III strategy, with 75% microalgal culture replacement by fresh medium, obtained similar content, productivity, and yield of lutein and protein as batch cultivation, demonstrating that it can be used for stable and continuous production. Fed-batch II strategy, feeding with 1/3 modified BG11 medium, achieved super-high lutein and protein yield (28.81 and 1592.77 mg/L, respectively), thus can be used for high-output production. Besides, two-stage strategy, combining light intensity shift and semi-batch cultivation, gained extremely high lutein and protein productivity (15.31 and 1080.41 mg/L/day, respectively), thereby is a good option for high-efficiency production. Moreover, the fed-batch II and two-stage strategy achieved high-quality lutein and protein, thus are promising for the co-production of lutein and protein in C. sorokiniana FZU60 for commercial application.

Comprehensive Utilization of Marine Microalgae for Enhanced Co-Production of Multiple Compounds.

Marine microalgae are regarded as potential feedstock because of their multiple valuable compounds, including lipids, pigments, carbohydrates, and proteins. Some of these compounds exhibit attractive bioactivities, such as carotenoids, ω-3 polyunsaturated fatty acids, polysaccharides, and peptides. However, the production cost of bioactive compounds is quite high, due to the low contents in marine microalgae. Comprehensive utilization of marine microalgae for multiple compounds production instead of the sole product can be an efficient way to increase the economic feasibility of bioactive compounds production and improve the production efficiency. This paper discusses the metabolic network of marine microalgal compounds, and indicates their interaction in biosynthesis pathways. Furthermore, potential applications of co-production of multiple compounds under various cultivation conditions by shifting metabolic flux are discussed, and cultivation strategies based on environmental and/or nutrient conditions are proposed to improve the co-production. Moreover, biorefinery techniques for the integral use of microalgal biomass are summarized. These techniques include the co-extraction of multiple bioactive compounds from marine microalgae by conventional methods, super/subcritical fluids, and ionic liquids, as well as direct utilization and biochemical or thermochemical conversion of microalgal residues. Overall, this review sheds light on the potential of the comprehensive utilization of marine microalgae for improving bioeconomy in practical industrial application.

Temperature elevation and acidification damage microstructure of abalone via expression change of crystal induction genes.

Ocean warming and acidification caused by global climate change interferes with the shell growth of mollusks. In abalone Haliotis discus hannai, the microstructural changes in the shell under stress are unclear, and the effect of thermal stress on biomineralization is unknown. The lack of gene information has also hampered the study of abalone biomineralization mechanisms. In this study, the microstructure of reconstructed shell in H. discus hannai was observed to determine the effects of thermal and acidification stress on shell growth. Three nacre protein genes, Hdh-AP7, Hdh-AP24, and Hdh-perlustrin, were characterized, and their expression pattern during shell repair was measured under thermal and acidification stress and compared with those of two known biomineralization-related genes, Hdh-AP-1 and Hdh-defensin. The stress resulted in aragonite plates with corroded or irregular microstructures. The gene expression of two nacre proteins (Hdh-AP7 and Hdh-AP24), which directly induce crystal formation, were more sensitive to thermal stress than to acidification, but the expression of the regulatory nacre protein (Hdh-perlustrin) and the two known genes (Hdh-AP-1 and Hdh-defensin), which are also related to immunity, showed an interlinked, complex pattern change. We concluded that high temperature and acidification damages the shell microstructure by disturbing the expression pattern of biomineralization-related genes.

Pilot-scale cultivation of Chlorella sorokiniana FZU60 with a mixotrophy/photoautotrophy two-stage strategy for efficient lutein production.

Chlorella sorokiniana FZU60, a lutein-enriching microalga, was cultivated in 50 L column photobioreactor to evaluate its potential for lutein production. Initial cell concentration, phosphate concentration and aeration rate were optimized, and results showed that optimal conditions of these three parameters were 0.10 g/L, 0.06 g/L and 0.02 vvm (2.5% CO2), respectively. In addition, a novel two-stage strategy was successfully developed, in which algae were firstly cultivated under fed-batch mixotrophic condition to achieve high biomass concentration, and then shifted to photoautotrophic condition for enhancing lutein accumulation. Moreover, dissolved oxygen was found to be an efficient indicator of acetate depletion in fed-batch stage. The obtained lutein content, production and productivity reached 9.51 mg/g, 33.55 mg/L and 4.67 mg/L/d, respectively, which were greater than those reported in other pilot-scale studies. This proposed strategy provided a cost-effective approach for high-efficient microalgae-based lutein production at pilot-scale, indicating great potential for commercial production.

Characteristics of microbial eukaryotic community recovery in eutrophic water by using ecological floating beds.

Ecological floating beds can rapidly remove nutrients (nitrogen and phosphorus) from eutrophic water, but we still know little about whether this process can simultaneously recover microbial eukaryotic communities. To fill this gap, planktonic microbial eukaryotic communities were investigated using 18S rRNA high-throughput gene sequencing during nutrient removal by floating beds of Canna indica L. We found that nutrient concentrations were high in both the control and treatment groups during period 1 (days 0-5) but rapidly decreased in the treatment group during period 2 (days 6-9) and period 3 (days 10-18). However, the microbial eukaryotic species richness and community compositions were similar between the control and treatment groups during periods 1 and 2 but showed small differences during period 3. The microbial eukaryotic co-occurrence networks between the control and treatment groups also showed similar degree centrality and interconnected eukaryotic members. We found that some abundant fungi species significantly responded to nutrient variations, but a large number of abundant ciliates were insensitive to nutrient removal. Our findings suggest that ecological floating beds can rapidly remove nutrients in eutrophic waters but that it is difficult to quickly and simultaneously improve microbial eukaryotic communities. This result reveals the critical influence of nutrient pollution on aquatic ecosystems and therefore on long-term and comprehensive aquatic habitat restoration, as aquatic macrophyte recoveries should be conducted after nutrient controls have been implemented.

Cloning and characterization of a novel Lustrin A gene from Haliotis discus hannai.

Lustrin A is the first nacre protein with specific structure and amino acid residue content that was identified in abalone; since its identification, homologs have been found in several abalone species. In this study, we isolated and cloned the complete cDNA of Lustrin A from Haliotis discus hannai, which was named Hdh-Lustrin A. Hdh-Lustrin A has characteristic cysteine- and proline-rich domains, glycine- and serine-rich domains, and a whey acidic protein (WAP)-like C-terminus. The cysteine- and proline-rich domains showed internal similarity repeats that arrayed in gene coding region, and the phylogenetic tree of these repeats indicated that the similarity of structural repetitive unit components in different abalone species, reflecting their evolutionary distance. A tissue distribution analysis showed that the mRNA level of Hdh-Lustrin A has tissue-specific expression in mantle. Under lipopolysaccharide (LPS) challenge, Hdh-Lustrin A showed a significantly increase, while it showed a more complex pattern with two peaks in the process of shell regeneration. Moreover, acidification and warming raised the expression level of Hdh-Lustrin A in shell regeneration in two different manners; acidification raised the gene expression in quick response, in contrast the long run in warming treatment. Similar pattern also has been detected in immune reaction and the thermal treatments. These results suggest that the Hdh-Lustrin A is a nacre protein, which can be distinguished by its cysteine- and proline-rich domain. It involves in shell regeneration and innate immunity in abalone, and its expression pattern during shell regeneration can be disrupted by physicochemical properties of the environment.

Biorefining and the Functional Properties of Proteins from Lipid and Pigment Extract Residue of Chlorella pyrenoidosa.

Microalgae are considered as excellent candidates for bioactive compounds, yet microalgal residues remaining after the extraction of one or two compounds are usually discarded, which is not economical. This study demonstrates the alkaline extraction of proteins from Chlorella pyrenoidosa residue after lipid and pigment extractions, and their functional properties. Single-factor experiments and response surface methodology were used to obtain the optimal conditions for protein extraction. Based on our results, a maximum protein yield of 722.70 mg/g, was obtained under the following extraction conditions: sodium hydroxide concentration 7.90%, extraction temperature 70.00 °C, extraction time 34.80 min, and microalgal residue concentration 8.20 mg/mL. The molecular weight of microalgal residue protein isolate (MRPI) was mainly distributed at the regions of 0.18-0.50 kDa, 0.50-1.50 kDa, and 1.50-5.00 kDa. The essential amino acid content was greater than the values recommended by FAO/WHO standards; a high essential amino acid index value (1.49) was another good indication that MRPI is suitable for human consumption. Moreover, MRPI exhibited excellent emulsifying properties and antioxidant activity, which suggests it may be useful as an emulsifying agent and antioxidant. These findings could improve the extraction methods of functional protein from microalgal residue and add value to microalgae-based bioactive compound production processes.

Bioprocess operation strategies with mixotrophy/photoinduction to enhance lutein production of microalga Chlorella sorokiniana FZU60.

This study isolated and identified the lutein-enriching microalga Chlorella sorokiniana FZU60. Different types of media and concentrations of sodium acetate and nitrate were evaluated to improve mixotrophic growth and lutein production. Highest lutein content, production, and productivity were obtained in BG11 medium with 1 g/L acetate and 0.75 g/L nitrate. Additionally, pulse feeding with 1 g/L acetate every 48 h led to the alternation between mixotrophy and photoinduction, resulting in a lutein production of 33.6 mg/L. Most notably, excellent lutein content (9.57 mg/g) and productivity (11.57 mg/L/d) were obtained using a new multi-operation integrated strategy, and the achieved levels exceed those reported in most related studies. This work demonstrates the synergistic integration of simple and effective strategies for the enhancement of lutein production in the indigenous microalga C. sorokiniana FZU60 and provides new insight into the highly efficient microalgae-based lutein production.

Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles.

The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 µmol/m2/s. Further increase in light intensity to 750 µmol/m2/s enhanced the biomass productivity to 1821.5 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.

Strategies related to light quality and temperature to improve lutein production of marine microalga Chlamydomonas sp.

The marine microalga Chlamydomonas sp. JSC4 was examined for its potential as a lutein producer. Environmental conditions, including light quality, temperature and light wavelength mixing ratio, were individually altered to enhance the cell growth rate and lutein production in strain JSC4. Results showed that optimal cell growth was obtained under white light and a temperature of 35 °C, while the optimal lutein content was obtained under blue light and a lower temperature of 20-25 °C. The best lutein production occurred when using a mixing ratio of 3:1 (white light: blue light). Strategies related to light quality and temperature (namely, temperature-gradient and two-stage strategies) were then used to further improve lutein production. Among them, the two-stage strategy proved to be effective markedly improving lutein content from 2.52 to 4.24 mg/g and resulting in the highest lutein productivity of 3.25 mg/L/day.

Manipulating Nutritional Conditions and Salinity-Gradient Stress for Enhanced Lutein Production in Marine Microalga Chlamydomonas sp.

Marine microalgae has great potential for lutein production with the advantage of saving fresh water resource. Thus, marine microalga Chlamydomonas sp. JSC4 is investigated as a potential lutein producer in this study. The medium types, nitrate-N and sea salt concentration are individually investigated to promote the cell growth rate and lutein production of JSC4. In Modified Bold Basal 3N medium, cell growth and lutein content are optimal at the nitrate-N concentration of 1000 mg L-1 and sea salt concentration of 2%. In addition, an innovative salinity-gradient strategy is operated to dramatically enhance biomass productivity (560 mg/L/d) and lutein content (3.42 mg g-1 ), resulting in the optimal lutein productivity (1.92 mg/L/d). Overall, this study clearly demonstrates that salinity is a significant inducer of lutein accumulation by strain JSC4 and that lutein production can be successfully optimized using the salinity-gradient strategy, which is beneficial for the outdoor large-scale lutein production in the future.