Effect of cell disruption methods on the extraction of bioactive metabolites from microalgal biomass.

Microalgae synthesize a variety of potentially high-value compounds. Due to their robust cell wall, cell disruption is necessary to improve extraction of these compounds. While cell disruption methods have been optimized for lipid and protein extraction, there are limited studies for other bioactive compounds. The present study investigated the effect of freeze-drying combined with sonication or ball-milling on the extraction of antioxidant and plant biostimulating compounds from Chlorella sp., Chlorella vulgaris and Scenedesmus acutus. Both cell disruption methods resulted in higher extract yields from the biomass compared to freeze-dried biomass using 50% methanol as a solvent. Antioxidant activity of Chlorella extracts was generally higher than freeze-dried extracts based on the diphenylpicrylhydrazyl (DPPH) and ß-carotene linoleic acid assays. However, the effectiveness of each treatment varied between microalgae strains. Sonication resulted in the highest antioxidant activity in Chlorella sp. extracts. Ball-milling gave the best results for C. vulgaris extracts in the DPPH assay. Both cell disruption methods decreased antioxidant activity in S. acutus extracts. Plant biostimulating activity was tested using the mung bean rooting assay. Damaging the membrane by freeze-drying was sufficient to release the active compounds using water extracts. In contrast, both cell disruption methods negatively affected the biological activity of the extracts. These results indicate that bioactive compounds in microalgae are sensitive to post-harvest processes and their biological activity can be negatively affected by cell disruption methods. Care must be taken to not only optimize yield but to also preserve the biological activity of the target compounds.

Dietary Scenedesmus ovalternus improves disease resistance of overwintering gibel carp (Carassius gibelio) by alleviating toll-like receptor signaling activation.

This study was conducted to investigate the effect of dietary Scenedesmus ovalternus on the growth and disease resistance of gibel carp (Carassius gibelio) during overwintering. Gibel carp (initial body weight: 90.39 ± 0.33 g) were fed with diets containing 0% or 4% Scenedesmus ovalternus (DS0 and DS4) for 4 weeks during the early overwintering period, and then all fish were left unfed during the late overwintering period. A bacterial challenge test using Aeromonas hydrophila was subsequently conducted. The 4% Scenedesmus ovalternus diet had no effect on the growth of gibel carp (P > 0.05), but did improve the survival rate after the challenge (P ≤ 0.05). In the DS0 group, the bacterial challenge decreased the contents of complement 3 (C3), immunoglobulin M (IgM), interleukin 2 (IL2) and tumor necrosis factor α (TNFα) in fish (P < 0.05); in the DS4 group, the challenge increased total antioxidant capacity (T-AOC) and myeloperoxidase (MPO) activity but decreased IL2 and TNFα contents (P < 0.05). The activities of MPO and contents of C3, IgM and TNFα were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P < 0.05). Compared to pre challenge, the expression levels of toll like receptor 2 (TLR2), toll like receptor 3 (TLR3), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon ß (TRIF), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), transforming growth factor ß (TGFß), interleukin 1ß (IL1ß), tumor necrosis factor α1 (TNFα1) and interleukin 10 (IL10) in the head kidney of gibel carp were induced after challenge (P < 0.05). Gibel carp fed the DS4 diet showed lower expression of TGFß in head kidney before the challenge and lower expression of TLR2, TLR3, TLR4, TIRAP, TRIF, IκBα, TNFα1, IL10 and TGFß after the challenge than that fed the DS0 diet (P < 0.05). Overall, Scenedesmus ovalternus supplement enhanced the resistances of gibel carp against A. hydrophila after overwintering via the TLR signaling pathway.

Supercritical Fluid Extraction of Lutein from Scenedesmus almeriensis.

Lutein has several benefits for human health, playing an important role in the prevention of age-related macular degeneration (AMD), cataracts, amelioration of the first stages of atherosclerosis, and some types of cancer. In this work, the Scenedesmus almeriensis microalga was used as a natural source for the supercritical fluid (SF) extraction of lutein. For this purpose, the optimization of the main parameters affecting the extraction, such as biomass pre-treatment, temperature, pressure, and carbon dioxide (CO2) flow rate, was performed. In the first stage, the effect of mechanical pre-treatment (diatomaceous earth (DE) and biomass mixing in the range 0.25-1 DE/biomass; grinding speed varying between 0 and 600 rpm, and pre-treatment time changing from 2.5 to 10 min), was evaluated on lutein extraction efficiency. In the second stage, the influence of SF-CO2 extraction parameters such as pressure (25-55 MPa), temperature (50 and 65 °C), and CO2 flow rate (7.24 and 14.48 g/min) on lutein recovery and purity was investigated. The results demonstrated that by increasing temperature, pressure, and CO2 flow rate lutein recovery and purity were improved. The maximum lutein recovery (~98%) with purity of ~34% was achieved operating at 65 °C and 55 MPa with a CO2 flow rate of 14.48 g/min. Therefore, optimum conditions could be useful in food industries for lutein supplementation in food products.

Carbohydrates and polyphenolics of extracts from genetically altered plant acts as catalysts for in vitro synthesis of silver nanoparticle.

Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement in bio-nanotechnology and has been successfully attempted in nearly 41 plant species. However, an established model plant system for systematically unraveling the biochemical components required for silver nanoparticles production is lacking. Here we used Arabidopsis thaliana as the model plant for silver nanoparticles biosynthesis in vitro. Employing biochemical, spectroscopic methods, selected mutants and over-expressor plants of Arabidopsis involved in pleotropic functions and sugar homeostasis, we show that carbohydrates, polyphenolics and glyco-proteins are essential components which stimulated silver nanoparticles synthesis. Using molecular genetics as a tool, our data enforces the requirement of sugar conjugated proteins as essentials for AgNPs synthesis over protein alone. Additionally, a comparative analysis of AgNPs synthesis using the aqueous extracts of some of the plant species found in a brackish water ecosystem (Gracilaria, Potamogeton, Enteromorpha and Scendesmus) were explored. Plant extract of Potamogeton showed the highest potential of nanoparticles production comparable to that of Arabidopsis among the species tested. Silver nanoparticles production in the model plant Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemical pathways and components in detail for its synthesis.

Influences of carbon and nitrogen sources and metal ions on the heterotrophic culture of Scenedesmus sp. LX1.

In this study, the influences of organic carbon sources (OCS, including xylose, glucose, maltose, sucrose, and starch) and inorganic and organic nitrogen sources (INS, including ammonia chloride and sodium nitrate; ONS, including arginine, alanine, proline, and valine) and metal ions (including Na+, K+, Mn2+, Zn2+ and Cu2+) on the growth, lipid accumulation, and nitrogen and phosphorus (N&P) removal capabilities of oleaginous Scenedesmus sp. LX1 under heterotrophic conditions were investigated. The results showed that glucose was the only OCS for Scenedesmus sp. LX1 to grow well with specific growth rate of 0.935 days-1, maximum biomass of 1.72 g L-1, and largest removal rates of N&P and organic carbon reaching 72.228%, 93.034%, and 19.208%, respectively. After 11 days of cultivation, the maximal biomass reached in the group with starch or glucose while maximal lipid and triacylglycerol (TAG) yields reached in the groups with maltose and sucrose, respectively. Sodium nitrate was best nitrogen source as the largest algal density, maximal yields of lipids and TAGs, and highest N&P removal rates reached up to 1.105 × 107 cells·mL-1, 196.70 mg L-1, 5.19 mg L-1, 89.61% and 100%, respectively. Scenedesmus sp. LX1 was found to have great tolerance to Na+, K+, Mn2+, and Zn2+ while 0.5 mg L-1 Cu2+ had a strong inhibition on growth and N&P removal rate of Scenedesmus sp. LX1. Concentration increasing of five metal ions all caused the yield increases of microalgal lipid and TAGs. Graphical abstract.

Evaluation of antioxidant and antibacterial capacity of green microalgae Scenedesmus subspicatus.

Microalgae are considered one of the most promising raw materials for the development of high value products for pharmaceuticals, nutraceuticals, and cosmetic industries, as well as being potential sources of protein, vitamins, and minerals for human consumption. Hence, the present research focuses extraction of antioxidant and antimicrobial compounds from Scenedesmus subspicatus using solvents of different polarities. Different solvents such as ethanol, methanol, butanol, acetone, dimethyl sulfoxide, and water were used to extract compounds from the green microalgae S. subspicatus and then they were examined for phytochemical screening, antioxidant activity, and antimicrobial properties. In vitro free radical quenching and total antioxidant activity of extracts were investigated with 1,1-diphenyl-2-picryl hydrazyl and compared with catequin and gallic acid as positive controls. The antimicrobial activity was evaluated in gram-negative and gram-positive bacteria. Aqueous extracts and dimethyl sulfoxide presented better performance in phytochemical analysis. This result showed consistency in the sequential tests. The antioxidant activity was also better using the two solvents cited above. The extracts acetone, water, and dimethyl sulfoxide showed ability to inhibit the growth of Bacillus subtilis. However, only dimethyl sulfoxide inhibited the growth of Klebsiella pneumoniae and Escherichia coli. Use of the aqueous extract, proven its effectiveness, is an economic protocol and avoids the use of toxic substances.

Protective effects of an extract of the freshwater microalga Scenedesmus rubescens on UV-irradiated skin cells.

BACKGROUND: Skin ageing results from intrinsic but also extrinsic factors of which UV irradiation is a main cause. It is hence of interest to have means to protect skin from UV irradiation-induced damage. We selected an extract of the freshwater microalga Scenedesmus rubescens and assessed its potential to protect skin from photoageing caused by UV irradiation. METHODS: Skin cells in vitro and ex vivo were analysed for markers of UV irradiation-induced photodamage such as decreased viability, decreased collagen content, hyperpigmentation and sunburn cells. RESULTS: We found that a dry extract of the microalga Scenedesmus rubescens was able to suppress cellular signs of ageing induced by UV irradiation. It enhanced dermal fibroblast viability, rescued dermal collagen content, inhibited the formation of sunburn cells and inhibited tyrosinase activity. CONCLUSION: An extract of Scenedesmus rubescens showed broad activity against markers of UV irradiation-induced cutaneous ageing. It may therefore be used as a preventive or regenerative agent for anti-ageing strategies.

Determining variables that influence the phosphorus content of waste stabilization pond algae.

Waste stabilization ponds (WSP) are one of the most common forms of wastewater treatment for smaller communities globally, but have poor phosphorus removal. It is known that WSP algae can accumulate polyphosphate within their cells in excess of that needed for cell function. If polyphosphate accumulation could be triggered at the higher range of WSP cell concentrations, phosphorus removal from domestic wastewater could be significantly improved. However, this phenomenon is sporadic and still not fully understood. With a view of building a fundamental understanding to underpin the engineering of a new phosphorus removal process, this paper examines eight previously untested variables that may influence the cellular phosphorus content of WSP biomass. Although calcium, magnesium, and potassium are key constituents of polyphosphate granules, the concentrations tested were not limiting to polyphosphate accumulation. While literature also pointed to inoculum characteristics as potentially having an impact, no significance was found in this research. Conversely, three important new triggers where identified that significantly (90% confidence) affected the cellular phosphorus content of WSP biomass. An increase in cellular phosphorus content was triggered by decreasing the organic load, or allowing the pH to increase as compared to pH control. By contrast, the presence of mixing decreased the phosphorus content of the WSP biomass.

Utilization of secondary-treated wastewater for the production of freshwater microalgae.

In this work, we studied the potential use of secondary-treated wastewater as nutrient source in the production of freshwater microalgae strains. Experiments were performed indoors in a semicontinuous mode, at 0.3 day(-1), simulating outdoor conditions. We demonstrated that all the tested strains can be produced by using only secondary-treated wastewater as the nutrient source. The utilization of secondary-treated wastewater imposes nutrient-limiting conditions, with maximal biomass productivity dropping to 0.5 g l(-1) day(-1) and modifies the biochemical composition of the biomass by increasing the amount of lipids and carbohydrates while reducing the biomass protein content. We measured fatty acid content and productivity of up to 25 %d.wt. and 110 mg l(-1) day(-1), respectively. We demonstrated that all the tested strains were capable of completely removing the nitrogen and phosphorus contained in the secondary-treated wastewater, and while the use of this effluent reduced the cells' photosynthetic efficiency, the nitrogen and phosphorus coefficient yield increased. Muriellopsis sp. and S. subpicatus were selected as the most promising strains for outdoor production using secondary-treated wastewater as the culture medium; this was not only because of their high productivity but also their photosynthetic efficiency, of up to 2.5 %, along with nutrient coefficient yields of up to 96 gbiomass gN (-1) and 166 gbiomass gP (-1). Coupling microalgae production processes to tertiary treatment in wastewater treatment plants make it possible to recover nutrients contained in the water and to produce valuable biomass, especially where nutrient removal is required prior to wastewater discharge.

Improvement of stability and carotenoids fraction of virgin olive oils by addition of microalgae Scenedesmus almeriensis extracts.

Humans are not capable of synthesizing carotenoids de novo and thus, their presence in human tissues is entirely of dietary origin. Consumption of essential carotenoids is reduced due to the lower intake of fruits and vegetables. Microalgae are a good source of carotenoids that can be exploited. In the present work, carotenoids rich extracts from Scenedesmus almeriensis were added to extra-virgin olive oils at different concentrations (0.1 and 0.21 mg/mL) in order to enhance the consumption of these bioactives. Extracts brought changes in olive oils color, turning them orange-reddish. Quality of olive oils was improved, since peroxidation was inhibited. Olive oils fatty acids and tocopherols were not affected. ß-carotene and lutein contents increase considerably, as well as oxidative stability, improving olive oils shelf-life and nutritional value. Inclusion of S. almeriensis extracts is a good strategy to improve and enhance the consumption of carotenoids, since olive oil consumption is increasing.

Simultaneous remediation of nutrients from liquid anaerobic digestate and municipal wastewater by the microalga Scenedesmus sp. AMDD grown in continuous chemostats.

AIMS: The primary aim of this study was to investigate the capacity of a microalga, Scenedesmus sp. AMDD, to remediate nutrients from municipal wastewater, either as the sole nutrient source or after blending with wastewater obtained from the anaerobic digestion of swine manure. A complimentary aim was to study and define the effects of these wastewaters on microalgal growth, biomass productivity and composition which have important implications for a commercial biofuels production system. METHODS AND RESULTS: A microalga, Scenedesmus sp. AMDD, was grown in continuous chemostats in municipal wastewater or wastewater supplemented with 1·6× or 2·4× higher levels of nitrogen (N) obtained through supplementation with anaerobic digestates. Biomass productivity increased with increasing nutrient supplementation, but was limited by light at high cell densities. Cellular quotas of carbon (C), nitrogen and phosphorus (P) all increased in direct proportion to their concentrations in the combined wastewaters. At higher cell densities, total carbohydrate decreased while protein increased. Fatty acid content remained relatively constant. Under high nutrient levels, the fatty acid profiles contained a higher concentration of polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Chlorophyll a was 2·5 times greater in the treatment of greatest nutrient supplementation compared to the treatment with the least. Ammonium (NH4(+)) and phosphate (PO4(3-)) were completely removed by algal growth in all treatments and with maximal removal rates of 41·2 mg N l(-1) d(-1) and 6·7 mg P l(-1) d(-1) observed in wastewater amended with 2·4× higher N level. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is the first to report stable, long-term continuous algal growth and productivity obtained by combining wastewaters of different sources. The study is supported by detailed analyses of the composition of the cultivated biomass and links composition to the nutrient and light availabilities in the cultures. Simultaneous remediation of these wastes by algal growth is discussed as a strategy for the valorization of the biomass.

Digestate color and light intensity affect nutrient removal and competition phenomena in a microalgal-bacterial ecosystem.

During anaerobic digestion, nutrients are mineralized and may require post-treatment for optimum valorization. The cultivation of autotrophic microalgae using the digestate supernatant is a promising solution; however the dark color of the influent poses a serious problem. First, the color of the digestates was studied and the results obtained using three different digestates demonstrated a strong heterogeneity although their color remained rather constant over time. The digestates absorbed light over the whole visible spectrum and remained colored even after a ten-fold dilution. Secondly, the impact of light and of substrate color on the growth of Scenedesmus sp. and on nitrogen removal were assessed. These experiments led to the construction of a model for predicting the impact of influent color and light intensity on N removal. Maximum N removal (8.5 mgN- [Formula: see text]  L(-1) d(-1)) was observed with an initial optical density of 0.221 and 244 µmolE m(-)² s(-1) light and the model allows to determine N removal between 15.9 and 22.7 mgN- [Formula: see text]  L(-1) d(-1) in real conditions according to the dilution level of the influent and related color. Changes in the microalgae community were monitored and revealed the advantage of Chlorella over Scenedesmus under light-limitation. Additionally microalgae outcompeted nitrifying bacteria and experiments showed how microalgae become better competitors for nutrients when phosphorus is limiting. Furthermore, nitrification was limited by microalgae growth, even when P was not limiting.

A comparative study of bio-oils from pyrolysis of microalgae and oil seed waste in a fluidized bed.

The pyrolysis of Scenedesmus sp. and Jatropha seedshell cake (JSC) was investigated under similar operating condition in a fluidized bed reactor for comparison of pyrolytic behaviors from different species of lipids-containing biomass. Microalgae showed a narrower main peak in differential thermogravimetric curve compared to JSC due to different constituents. Pyrolysis liquid yields were similar; liquid's oil proportion of microalgae is higher than JSC. Microalgae bio-oil was characterized by similar carbon and hydrogen contents and higher H/C and O/C molar ratios compared to JSC due to compositional difference. The pyrolytic oils from microalgae and JSC contained more oxygen and nitrogen and less sulfur than petroleum and palm oils. The pyrolytic oils showed high yields of fatty oxygenates and nitrogenous compounds. The microalgae bio-oil features in high concentrations of aliphatic compounds, fatty acid alkyl ester, alcohols and nitriles. Microalgae showed potentials for alternative feedstock for green diesel, and commodity and valuable chemicals.

The good, the bad and the plenty: interactive effects of food quality and quantity on the growth of different Daphnia species.

Effects of food quality and quantity on consumers are neither independent nor interchangeable. Although consumer growth and reproduction show strong variation in relation to both food quality and quantity, the effects of food quality or food quantity have usually been studied in isolation. In two experiments, we studied the growth and reproduction in three filter-feeding freshwater zooplankton species, i.e. Daphnia galeata x hyalina, D. pulicaria and D. magna, on their algal food (Scenedesmus obliquus), varying in carbon to phosphorus (C∶P) ratios and quantities (concentrations). In the first experiment, we found a strong positive effect of the phosphorus content of food on growth of Daphnia, both in their early and late juvenile development. Variation in the relationship between the P-content of animals and their growth rate reflected interspecific differences in nutrient requirements. Although growth rates typically decreased as development neared maturation, this did not affect these species-specific couplings between growth rate and Daphnia P-content. In the second experiment, we examined the effects of food quality on Daphnia growth at different levels of food quantity. With the same decrease in P-content of food, species with higher estimated P-content at zero growth showed a larger increase in threshold food concentrations (i.e. food concentration sufficient to meet metabolic requirements but not growth). These results suggest that physiological processes such as maintenance and growth may in combination explain effects of food quality and quantity on consumers. Our study shows that differences in response to variation in food quality and quantity exist between species. As a consequence, species-specific effects of food quality on consumer growth will also determine how species deal with varying food levels, which has implications for resource-consumer interactions.

Thermochemical conversion of raw and defatted algal biomass via hydrothermal liquefaction and slow pyrolysis.

Thermochemical conversion is a promising route for recovering energy from algal biomass. Two thermochemical processes, hydrothermal liquefaction (HTL: 300 °C and 10-12 MPa) and slow pyrolysis (heated to 450 °C at a rate of 50 °C/min), were used to produce bio-oils from Scenedesmus (raw and defatted) and Spirulina biomass that were compared against Illinois shale oil. Although both thermochemical conversion routes produced energy dense bio-oil (35-37 MJ/kg) that approached shale oil (41 MJ/kg), bio-oil yields (24-45%) and physico-chemical characteristics were highly influenced by conversion route and feedstock selection. Sharp differences were observed in the mean bio-oil molecular weight (pyrolysis 280-360 Da; HTL 700-1330 Da) and the percentage of low boiling compounds (bp<400 °C) (pyrolysis 62-66%; HTL 45-54%). Analysis of the energy consumption ratio (ECR) also revealed that for wet algal biomass (80% moisture content), HTL is more favorable (ECR 0.44-0.63) than pyrolysis (ECR 0.92-1.24) due to required water volatilization in the latter technique.

Stability of carotenoids in Scenedesmus almeriensis biomass and extracts under various storage conditions.

Scenedesmus almeriensis biomass is a source of carotenoids, particularly lutein, and is considered to be promising as an alternative source to marigold. One key question concerning alternative sources of lutein is the loss of carotenoids that takes place between harvesting and processing, which in the case of marigold is frequently up to 50%. The work described here involved a study into the stability of the main carotenoids (lutein, violaxanthin, and beta-carotene), as well as other components, under different storage conditions. The experiments were carried out with biomass in three forms: frozen, freeze-dried, and spray-dried. The stability of extracts of Scenedesmus biomass in acetone and olive oil was also studied. The results show that the most important factor in retaining carotenoids is a low temperature. At -18 degrees C the loss of carotenoids was negligible after the storage period, regardless of the biomass form used (frozen, freeze-dried, or spray-dried). On the other hand, the carotenoid content and fatty acid profile was increasingly affected with increasing temperature. However, the protein content is unaffected by storage conditions.

Algal-based immobilization process to treat the effluent from a secondary wastewater treatment plant (WWTP).

Algal-based immobilization process was applied to treat the effluent from a secondary wastewater treatment plant. Batch test proved that algae could attach onto fiber-bundle carrier in 7 days, and then the algal-based immobilization reactor could reduce TN (total nitrogen) and TP (total phosphorus) significantly within 48 h. Based on the above investigations, the hydraulic retention time (HRT) of the algal-based immobilization reactor in continuous operation mode was determined to be 2 days. During the 91 days of experiment on the treating secondary effluent of Guang-Rao wastewater treatment plant, it was found that the fiber-bundle carrier could collect the heterobacteria and nitrifying bacteria gradually, and thus improved the COD removal efficiency and nitrification performance step by step. Results of the continuous operation indicated that the final effluent could meet the Chinese National First A-level Sewage Discharge Standard when the algal-based immobilization reactor reached steady state.

Dietary assimilation and elimination of Cd, Se, and Zn by Daphnia magna at different metal concentrations.

This study examines the influences of Cd, Se, and Zn concentrations in ambient water on the assimilation, elimination, and release budget of metals, and the clearance rate of Daphnia magna through ingestion of phytoplankton diets. With increasing ambient metal concentration over two to three orders of magnitude, the assimilation efficiencies (AEs) of Cd and Se from two green algal species, Scenedesmus obliquus and Chlamydomonas reinhardtii, decreased significantly. In contrast, the Zn AE from ingested food, which typically was low (<7%), was not impacted by the difference in Zn or Cd concentrations in ingested cells. The Cd AE also was reduced significantly by a high Zn concentration in ingested algae, perhaps due to the competitive assimilation between the two metals. Metal elimination was determined after the daphnids were exposed to the radiolabeled diets for 3 d. The elimination rate constant was not dependent on the metal concentration in ingested food. Similarly, the routes of metal elimination (excretion, egestion, molting, and neonate production) were less influenced by the metal concentration. The rapid elimination of Se and Zn may be attributed to the transfer of these metals from mother to offspring. Except at the highest Cd (917 nM) and Se (1,333 nM) concentrations, there was no significant effect of metal concentration on the clearance rate of the animals. Consequently, the metal assimilation in D. magna may play an important role in the regulation of Cd and Se influx into the animals, whereas the regulation of the metal tissue burden in the animals is unlikely to be achieved through the metal elimination. The trophic transfer factors (TTF) of Cd and Se were related inversely to the metal concentration in the water, whereas the TTF of Zn was less variable at different Zn concentrations. The change of TTF with metal concentration will have important implication for ecotoxicity testing using D. magna, especially when the dietary exposure is incorporated.