Antimicrobial and Antioxidant Potential of Scenedesmus obliquus Microalgae in the Context of Integral Biorefinery Concept.

Small-scale photobioreactors (PBRs) in the inoculum stage were designed with internal (red or green) and external white LED light as an initial step of a larger-scale installation aimed at fulfilling the integral biorefinery concept for maximum utilization of microalgal biomass in a multifunctional laboratory. The specific growth rate of Scenedesmus obliquus (Turpin) Kützing biomass for given cultural conditions was analyzed by using MAPLE software. For the determination of total polyphenols, flavonoids, chlorophyll "a" and "b", carotenoids and lipids, UHPLC-HRMS, ISO-20776/1, ISO-10993-5 and CUPRAC tests were carried out. Under red light growing, a higher content of polyphenols was found, while the green light favoured the flavonoid accumulation in the biomass. Chlorophylls, carotenoids and lipids were in the same order of magnitude in both samples. The dichloromethane extracts obtained from the biomass of each PBR synergistically potentiated at low concentrations (0.01-0.05 mg/mL) the antibacterial activity of penicillin, fluoroquinolones or oregano essential oil against the selected food-borne pathogens (Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) without showing any in vitro cytotoxicity. Both extracts exhibited good cupric ion-reducing antioxidant capacity at concentrations above 0.042-0.08 mg/mL. The UHPLC-HRMS analysis revealed that both extracts contained long chain fatty acids and carotenoids thus explaining their antibacterial and antioxidant potential. The applied engineering approach showed a great potential to modify microalgae metabolism for the synthesis of target compounds by S. obliquus with capacity for the development of health-promoting nutraceuticals for poultry farming.

Phenoplate: An innovative method for assessing interacting effects of temperature and light on non-photochemical quenching in microalgae under chemical stress.

Rapid light curves are one of the most widely used methods for assessing the physiological state of photosynthetic organisms. While the method has been applied in a range of physiological studies over the last 20 years, little progress has been made in adapting it for the new age of multi-parametric phenotyping. In order to advance research that is aimed at evaluating the physiological impact of multiple factors, the Phenoplate was developed: a simultaneous assessment of temperature and light gradients. It was used to measure rapid light curves of three marine microalgae across a temperature gradient and altered phosphate availability. The results revealed that activation of photoprotective mechanisms occurred with high efficiency at lower temperatures, and relaxation of photoprotection was negatively impacted above a certain temperature threshold in Tetraselmis sp. It was observed that Thalassiosira pseudonana and Nannochloropsis oceanica exhibited two unique delayed non-photochemical quenching signatures: in combinations of low light with low temperature, and darkness with high temperature, respectively. These findings demonstrate that the Phenoplate approach can be used as a rapid and simple tool to gain insight into the photobiology of microalgae.

Thermo resistant antioxidants from photoautotrophic microorganisms: screening and characterization.

The demand for natural antioxidants to be used in food industry is increasing, as synthetic antioxidants are toxic and have high production costs. Specifically, food processing and preservation require antioxidants resistant to thermal sterilization processes. In this study, twenty-five strains among microalgae and cyanobacteria were screened as antioxidants producers. The species Enallax sp., Synechococcus bigranulatus and Galdieria sulphuraria showed the highest content of chlorophyll a and total carotenoids. In vitro stability and antioxidant activity of the ethanolic extracts were performed. The results revealed that pigments present in the extracts, obtained from the previously mentioned species, were stable at room temperature and exhibited in vitro free radical scavenging potential with IC50 values of 0.099 ± 0.001, 0.048 ± 0.001 and 0.13 ± 0.02 mg mL-1, respectively. Biocompatibility assay showed that the extracts were not toxic on immortalized cell lines. The antioxidant activity was also tested on a cell-based model by measuring intracellular ROS levels after sodium arsenite treatment. Noteworthy, extracts were able to exert the same protective effect, before and after the pasteurization process. Results clearly indicate the feasibility of obtaining biologically active and thermostable antioxidants from microalgae. Green solvents can be used to obtain thermo-resistant antioxidants from cyanobacteria and microalgae which can be used in the food industry. Thus, the substitution of synthetic pigments with natural ones is now practicable.

Microwave-Assisted Defibrillation of Microalgae.

The first production of defibrillated celluloses from microalgal biomass using acid-free, TEMPO-free and bleach-free hydrothermal microwave processing is reported. Two routes were explored: i. direct microwave process of native microalgae ("standard"), and ii. scCO2 pre-treatment followed by microwave processing. ScCO2 was investigated as it is commonly used to extract lipids and generates considerable quantities of spent algal biomass. Defibrillation was evidenced in both cases to afford cellulosic strands, which progressively decreased in their width and length as the microwave processing temperature increased from 160 °C to 220 °C. Lower temperatures revealed aspect ratios similar to microfibrillated cellulose whilst at the highest temperature (220 °C), a mixture of microfibrillated cellulose and nanocrystals were evidenced. XRD studies showed similar patterns to cellulose I but also some unresolved peaks. The crystallinity index (CrI), determined by XRD, increased with increasing microwave processing temperature. The water holding capacity (WHC) of all materials was approximately 4.5 g H2O/g sample. The materials were able to form partially stable hydrogels, but only with those processed above 200 °C and at a concentration of 3 wt% in water. This unique work provides a new set of materials with potential applications in the packaging, food, pharmaceutical and cosmetic industries.

Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging.

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.

The Effect of Abiotic Factors on Abundance and Photosynthetic Performance of Airborne Cyanobacteria and Microalgae Isolated from the Southern Baltic Sea Region.

Cyanobacteria and microalgae present in the aquatic or terrestrial environment may be emitted into the air and transported along with air masses over long distances. As a result of staying in the atmosphere, these organisms may develop a greater tolerance to stressful factors, but this topic is still relatively unknown. The main aim was to show an autecological characteristic of some airborne microalgae and cyanobacteria strains by a factorial laboratory experiment approach, including changes in irradiance, temperature, and salinity conditions. The additional purpose of this work was also to present part of the Culture Collection of Baltic Algae (CCBA) collection, which consists of airborne algae (AA) isolated from the atmospheric air of the southern Baltic Sea region. Altogether, 61 strains of airborne cyanobacteria and microalgae from the southern Baltic Sea region were isolated from May 2018 to August 2020. Selected microorganisms were tested in controlled laboratory conditions to identify their response to different irradiance (10-190 µmol photons m-2 s-1), temperature (13-23 °C), and salinity conditions (0-36 PSU). The highest numbers of cells (above 30 × 105 cell mL-1) were recorded for cyanobacterium Nostoc sp., and for diatoms Nitzschia sp., Amphora sp., and Halamphora sp. We found that for cyanobacterium Nostoc sp. as well as for green alga Coccomyxa sp. the maximum cell concentrations were recorded at the salinity of 0 PSU. Moreover, cyanobacteria Planktolyngbya contorta, Pseudanabaena catenata, Leptolyngbya foveolarum, Gloeocapsa sp., and Rivularia sp. were able to grow only at a salinity of 0 PSU. On the other hand, in the range of 16-24 PSU, the highest cell numbers of examined diatoms have been identified. Our research provided that deposited airborne microalgae and cyanobacteria showed full colonization potential. The present experiment suggests that the adaptive abilities of microorganisms, in particular those producing toxins, may contribute to the spread in the future. Thus, it may increase human exposure to their negative health effects. Any distinctive adaptations of the genera give them an additional competitive advantage and a greater chance for territorial expansion.

Effects of light-emitting diodes (LEDs) on lipid production of the aerial microalga Coccomyxa sp. KGU-D001 under liquid- and aerial-phase conditions.

Algal biofuels are a promising alternative to fossil fuels, but their widespread use is hindered by problems with mass production. Light-emitting diodes (LEDs) with specific light wavelengths could be used as an energy source for algal growth and lipid synthesis. In this study, the effects of light source on the biomass and lipid production of the aerial microalga Coccomyxa sp. KGU-D001 were evaluated using LEDs. The integration of two-phase cultures, including growth and lipid production under the stress of nitrate depletion, was assessed for efficient lipid production under liquid- or aerial-phase conditions. Different wavelengths of light (blue, green, and red) were tested under liquid- and aerial-phase conditions. Under aerial-phase culture, the fatty acid contents in biofilm reached 320 mg g DWC-1 with the red LEDs. In view of these findings, we describe a one-step culture method for growth and lipid accumulation in algal biofilm under aerial-phase culture with red LED irradiation. When Coccomyxa biofilm was cultured on wet cotton wool with BBM in a petri dish under the red LED, it was able to grow and accumulate lipids under the aerial-phase condition. Based on the results of this study, a potential method for a continuous biodiesel production system is proposed.

Scalable Cultivation of Engineered Cyanobacteria for Squalene Production from Industrial Flue Gas in a Closed Photobioreactor.

Economically feasible photosynthetic cultivation of microalgal and cyanobacterial strains is crucial for the biological conversion of CO2 and potential CO2 mitigation to challenge global warming. To overcome the economic barriers, the production of value-added chemicals was desired by compensating for the overall processing cost. Here, we engineered cyanobacteria for photosynthetic squalene production and cultivated them in a scalable photobioreactor using industrial flue gas. First, an inducer-free gene expression system was developed for the cyanobacteria to lower production const. Then, the recombinant cyanobacteria were cultivated in a closed photobioreactor (100 L) using flue gas (5% CO2) as the sole carbon source under natural sunlight as the only energy source. Seasonal light intensities and temperatures were analyzed along with cyanobacterial cell growth and squalene production in August and October 2019. As a result, the effective irradiation hours were the most critical factor for the large-scale cultivation of cyanobacteria. Thus, an automated photobioprocess system will be developed based on the regional light sources.

Crosstalk between sterol and neutral lipid metabolism in the alga Haematococcus pluvialis exposed to light stress.

The presence, biosynthesis and functional role of sterols in the green microalga Haematococcus pluvialis remain poorly understood. In this work we studied the effect of high-light (HL) stress on sterol synthesis in H. pluvialis UTEX 2505 cells. HL stress induced the synthesis of sterols in parallel with that of triacylglycerides (TAG), giving rise to the synthesis of cholesterol over that of phytosterols. Blockage of the carotenogenic 1-deoxy-D-xylulose 5-phosphate (MEP) pathway is shown to be involved in HL-induced sterol synthesis. In addition, high irradiance exposure induced MEP- and fatty acid (FA)-biosynthetic transcripts. The pharmacological inhibition of these pathways suggests a possible feedback regulation of sterol and FA homeostasis. Finally, both lipid classes proved crucial to the adequate photosynthetic performance of H. pluvialis grown under HL intensity stress. Our findings reveal new insights into H. pluvialis lipid metabolism that contribute to the development of value-added bioproducts from microalgae.

Bionic 3D printed corals.

Corals have evolved as optimized photon augmentation systems, leading to space-efficient microalgal growth and outstanding photosynthetic quantum efficiencies. Light attenuation due to algal self-shading is a key limiting factor for the upscaling of microalgal cultivation. Coral-inspired light management systems could overcome this limitation and facilitate scalable bioenergy and bioproduct generation. Here, we develop 3D printed bionic corals capable of growing microalgae with high spatial cell densities of up to 109 cells mL-1. The hybrid photosynthetic biomaterials are produced with a 3D bioprinting platform which mimics morphological features of living coral tissue and the underlying skeleton with micron resolution, including their optical and mechanical properties. The programmable synthetic microenvironment thus allows for replicating both structural and functional traits of the coral-algal symbiosis. Our work defines a class of bionic materials that is capable of interacting with living organisms and can be exploited for applied coral reef research and photobioreactor design.

Harnessing C/N balance of Chromochloris zofingiensis to overcome the potential conflict in microalgal production.

Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.

Quantitative viability detection for a single microalgae cell by two-level photoexcitation.

A novel method for quantitative detection of the viability of a single microalgae cell by two-level photoexcitation is proposed in this paper. This method overcomes the difficulty of traditional methods in determining the cell viability by a fixed standard under a single photoexcitation. It is experimentally confirmed that this method is not limited by the species, morphology, size and structure of microalgae cells. An evaluation criterion of universal applicability is presented for the assessment of cell viability based on the large amount of experimental data. To the best of our knowledge, this is the first time that the relative fluorescence yield ratio Fr has been used to characterize the viability of single microalgae cells during cell migration. By using the relative fluorescence yield ratio, this method does not require the intensity of the excitation light to be very low for the assessment of the fluorescence yield of a dark-adapted microalgae cell, nor to be very strong to reach the saturated light level to assess the maximum fluorescence yield. Therefore, this method greatly reduces the technical difficulties of developing a sensor device. Well balanced portability, accuracy and universal applicability make it suitable for on-site real-time detection.

Understanding how the collective behaviour of phytoflagellates is affected by light attenuation and diel vertical migration using individual-based modelling.

In this article, we investigate the question of the impact of the diel vertical migration (DVM) and the light attenuation by the cells on both spatial pattern and population dynamics of phytoflagellates. For these purposes, we performed a simulation study by using a spatially explicit individual-based model (IBM). The designed IBM includes 2 global mechanisms: cellular motion which is modelled through a stochastic differential equation and demographic process that is density and light intensity dependent. We showed that under no-DVM, for competitive environment, increasing the light absorption helps the creation of a strong oscillatory behaviour, that breeds aggregation-break up rhythm, which is beneficial. However, when we pass to DVM regime, it will be more advantageous for the cells to reduce their light absorption. In regards of DVM effect, we showed that it has other benefits, apart from the acquisition of resources, such as enhancing the attraction mechanism that promotes cooperation and also helping the creation of spatial voids that permit the penetration of the light. Also, we showed that the DVM reduces the predation rate, so we prove quantitatively that the DVM reduces, in general, the grazing losses. Also, we found that the DVM strategy depends on the impact of the competition and cooperation between the cells on the division and death rates. So, probably for the motile species, the local competition is not high in order to let the cells gain advantages from the DVM regarding their survivability that is explained by an annual cyclic behaviour under seasonal environment.

Kinetic characterization and modeling of a microalgae consortium isolated from landfill leachate under a high CO2 concentration in a bubble column photobioreactor

BACKGROUND: The determination of kinetic parameters and the development of mathematical models are of great interest to predict the growth of microalgae, the consumption of substrate and the design of photobioreactors focused on CO2 capture. However, most of the models in the literature have been developed for CO2 concentrations below 10%. RESULTS: A nonaxenic microalgal consortium was isolated from landfill leachate in order to study its kinetic behavior using a dynamic model. The model considered the CO2 mass transfer from the gas phase to the liquid phase and the effect of light intensity, assimilated nitrogen concentration, ammonium concentration and nitrate concentration. The proposed mathematical model was adjusted with 13 kinetic parameters and validated with a good fit obtained between experimental and simulated data. CONCLUSIONS: Good results were obtained, demonstrating the robustness of the proposed model. The assumption in the model of DIC inhibition in the ammonium and nitrate uptakes was correct, so this aspect should be considered when evaluating the kinetics with microalgae with high inlet CO2 concentrations.

CaCl2 pretreatment improves gamma toxicity tolerance in microalga Chlorella vulgaris.

The Chlorella vulgaris has been generally recognized as a promising microalgal model to study stress-related responses due to its ability to withstand against ionizing and non-ionizing radiation. The objective of the present study was to investigate the effect of CaCl2 pre-treatment at different concentrations on the responses of microalga C. vulgaris under gamma radiation toxicity. Changes in growth, physiological parameters and biochemical compositions of the algae pretreated with 0.17 (normal), 5, and 10 mM CaCl2 were analyzed under 300 Gy gamma irradiation and compared to those of gamma-free control. The results showed that parameters including specific growth rate, cell size, chlorophyll and protein contents, ascorbate peroxidase (APX), and superoxide dismutase (SOD) activity, Ferric Reducing Antioxidant Power (FRAP), and the ratios of nucleic acid to protein negatively affected by gamma irradiation. All these parameters, except for the ratios of nucleic acid to protein significantly increased in the algae when pretreated with a CaCl2 content higher than normal concentration. The analysis also showed that parameters including catalase activity, proline, and carotenoid content, the level of lipid peroxidation, and electrolyte leakage (EL) significantly increased under gamma irradiation but not affected significantly under different CaCl2 pre-treatments. Additionally, specific growth rate, chlorophyll a and protein content, APX and SOD activity, FRAP, lipid peroxidation, electrolyte leakage, and the ratios of nucleic acid to protein were the only parameters that significantly affected by the interaction of gamma toxicity and CaCl2 pretreatment. Overall, the results suggested that regardless of the CaCl2 effect, the algal cells responded to gamma radiation more efficiently by increasing proline, carotenoids content, and CAT activity. More important, it was concluded that calcium had an essential role in modifying the detrimental effect of gamma toxicity on the algae mainly by increasing the activity of ascorbate peroxidase and superoxide dismutase and maintaining the reducing antioxidant power (FRAP) of the cells at a high level.

Selenium Incorporation to Amino Acids in Chlorella Cultures Grown in Phototrophic and Heterotrophic Regimes.

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 µg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Reconstruction and analysis of a carbon-core metabolic network for Dunaliella salina.

BACKGROUND: The green microalga Dunaliella salina accumulates a high proportion of ß-carotene during abiotic stress conditions. To better understand the intracellular flux distribution leading to carotenoid accumulation, this work aimed at reconstructing a carbon core metabolic network for D. salina CCAP 19/18 based on the recently published nuclear genome and its validation with experimental observations and literature data. RESULTS: The reconstruction resulted in a network model with 221 reactions and 212 metabolites within three compartments: cytosol, chloroplast and mitochondrion. The network was implemented in the MATLAB toolbox CellNetAnalyzer and checked for feasibility. Furthermore, a flux balance analysis was carried out for different light and nutrient uptake rates. The comparison of the experimental knowledge with the model prediction revealed that the results of the stoichiometric network analysis are plausible and in good agreement with the observed behavior. Accordingly, our model provides an excellent tool for investigating the carbon core metabolism of D. salina. CONCLUSIONS: The reconstructed metabolic network of D. salina presented in this work is able to predict the biological behavior under light and nutrient stress and will lead to an improved process understanding for the optimized production of high-value products in microalgae.

Influence of light on the infection of Aureococcus anophagefferens CCMP 1984 by a "giant virus".

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980's, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 µmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 µmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur.

Enhancing production of microalgal biopigments through metabolic and genetic engineering.

The versatile use of biopigments in food, feed, cosmetic, pharmaceutical and analytical industries emphasized to find different and renewable sources of biopigments. Microalgae, including cyanobacteria, are becoming a potential candidate for pigment production as these have fast-growing ability, high pigment content, highly variable and also have "Generally recognized as safe" status. These algal groups are known to produce different metabolites that include hormones, vitamins, biopolythene and biochemicals. We discuss here the potential use of microalgal biopigments in our daily life as well as in food and cosmetic industries. Pigment like carotenoids has many health benefits such as antioxidant, anti-inflammatory properties and also provide photo-protection against UV radiation. This review details the effect of various abiotic and biotic factors such as temperature, light, nutrition on maximizing the pigment content in the microalgal cell. This review also highlights the potential of microalgae, whether in present native or engineered strain including the many metabolic strategies which are used or can be used to produce a higher amount of these valuable biopigments. Additionally, future challenges in the context of pigment production have also been discussed.

Computational fluid dynamics simulation in scaling-up of airlift photobioreactor for astaxanthin production.

The unicellular green microalga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin. Aiming to cultivate these microalgae with high astaxanthin efficiency, cultivations were scaled-up from 1000 mL bottle to 2 L and 8 L airlift photobioreactor using volumetric power consumption rate (W/m3) as scale up strategy. After cultivations, computational fluid dynamics (CFD) simulation was used to investigate the flow patterns, mixing efficiency and gas holdup profile within the 2 L photobioreactor. At the end, astaxanthin content was enhanced with increasing the cultivation volume and highest astaxanthin amount of 49.39 ± 1.64 mg/g cell was obtained in 8 L photobioreactor. Hydrodynamic characteristics of photobioreactor was simulated and gas holdup showed difference between the riser and the downcomer regions. Velocity profiles of air and medium had higher values inside the draft tube than obtained in downcomer region. However liquid circulation was achieved from draft tube to the downcomer, mixing was not provided effectively considering the turbulence kinetic energy. For the further research, some developments about column configuration, sparger diameter may be necessary to enhance the mixing characteristics.