Combined Dietary Administration of Chlorella fusca and Ethanol-Inactivated Vibrio proteolyticus Modulates Intestinal Microbiota and Gene Expression in Chelon labrosus.

The use of functional feeds in aquaculture is currently increasing. This study aimed to assess the combined impact of dietary green microalgae Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (CVP diet) on thick-lipped grey mullet (Chelon labrosus) juvenile fish. The effects on intestinal microbiota and the transcription of genes related to metabolism, stress, and the immune system were investigated after 90 days of feeding. Additionally, the fish were challenged with Aeromonas hydrophila and polyinosinic-polycytidylic acid (poly I:C) to evaluate the immune response. Microbiota analysis revealed no significant differences in alpha and beta diversity between the anterior and posterior intestinal sections of fish fed the control (CT) and CVP diets. The dominant genera varied between the groups; Pseudomonas and Brevinema were most abundant in the CVP group, whereas Brevinema, Cetobacterium, and Pseudomonas were predominant in the CT group. However, microbial functionality remained unaltered. Gene expression analysis indicated notable changes in hif3α, mhcII, abcb1, mx, and tnfα genes in different fish organs on the CVP diet. In the head kidney, gene expression variations were observed following challenges with A. hydrophila or poly I:C, with higher peak values seen in fish injected with poly I:C. Moreover, c3 mRNA levels were significantly up-regulated in the CVP group 72 h post-A. hydrophila challenge. To conclude, incorporating C. fusca with V. proteolyticus in C. labrosus diet affected the microbial species composition in the intestine while preserving its functionality. In terms of gene expression, the combined diet effectively regulated the transcription of stress and immune-related genes, suggesting potential enhancement of fish resistance against stress and infections.

Influence of pH and dissolved oxygen control strategies on the performance of pilot-scale microalgae raceways using fertilizer or wastewater as the nutrient source.

Dissolved oxygen concentration and pH are controllable and cost-effective variables that determine the success of microalgae-related processes. The present study compares different control strategies for pH and dissolved oxygen in pilot-scale microalgae production systems. Two 80 m2 raceway reactors were used, one operated with freshwater plus fertilizer and the other with wastewater as the nutrient source. Both were in semi-continuous mode at a fixed dilution rate of 0.2 day-1. A comparison between the classical On-Off and more advanced pH control strategies, such as PI and Event-based control, was performed, focusing on biomass productivity and the influence of all the process parameters on microalgae growth; "No control" of pH was also assayed. The results show that Event-based control was the best algorithm when using freshwater plus fertilizer. In contrast, no significant differences were observed using the different control strategies when wastewater was the nutrient source. These experiments were performed through selective control strategy, prioritizing pH over dissolved oxygen; however, it was demonstrated that they did not allow to achieve satisfactory dissolved oxygen removal results, especially for the fertilizer system. After modifying the gas diffuser configuration and improving the mass transfer, independent on-off strategies have been developed, permitting effective control of both variables and increasing productivity by up to 20% in both systems. Concluding, a detailed analysis of the energy demand for each strategy implemented in terms of gas consumption and gas flow to biomass ratio is provided.

Influence of culture media composition on the rheology of microalgae concentrates on a large scale.

The role of microalgae in the production of bioproducts and biofuels, along with their ability to provide a sustainable pathway for wastewater treatment, makes them promising alternatives to conventional processes. Nevertheless, large-scale downstream processing requires an understanding of biomass rheology that needs to be addressed further. This study aimed to characterize microalgal concentrates rheologically in different culture media. The presence of bacteria was quantified by photorespirometry and plate counting techniques. The culture medium was found to significantly influence viscosity, with primary wastewater exhibiting the highest viscosity and seawater plus pig slurry the lowest. The concentration of heterotrophic bacteria was directly related to the viscosity. Extracellular polysaccharides (EPS) in supernatant exhibited an inverse viscosity trend compared to biomass concentrates, with pig slurry cultures having higher concentrations. These findings emphasize the profound influence of culture medium and EPS on the rheology of microalgal biomass, underscoring the need for continued research aimed at facilitating and optimizing large-scale downstream processes within the framework of a circular economy and the attainment of the Sustainable Development Goals (6,8, and 12).

In Vitro and In Vivo Effects of Ulvan Polysaccharides from Ulva rigida.

One of the main bioactive compounds of interest from the Ulva species is the sulfated polysaccharide ulvan, which has recently attracted attention for its anticancer properties. This study investigated the cytotoxic activity of ulvan polysaccharides obtained from Ulva rigida in the following scenarios: (i) in vitro against healthy and carcinogenic cell lines (1064sk (human fibroblasts), HACAT (immortalized human keratinocytes), U-937 (a human leukemia cell line), G-361 (a human malignant melanoma), and HCT-116 (a colon cancer cell line)) and (ii) in vivo against zebrafish embryos. Ulvan exhibited cytotoxic effects on the three human cancer cell lines tested. However, only HCT-116 demonstrated sufficient sensitivity to this ulvan to make it relevant as a potential anticancer treatment, presenting an LC50 of 0.1 mg mL-1. The in vivo assay on the zebrafish embryos showed a linear relationship between the polysaccharide concentration and growth retardation at 7.8 hpf mL mg-1, with an LC50 of about 5.2 mg mL-1 at 48 hpf. At concentrations near the LC50, toxic effects, such as pericardial edema or chorion lysis, could be found in the experimental larvae. Our in vitro study supports the potential use of polysaccharides extracted from U. rigida as candidates for treating human colon cancer. However, the in vivo assay on zebrafish indicated that the potential use of ulvan as a promising, safe compound should be limited to specific concentrations below 0.001 mg mL-1 since it revealed side effects on the embryonic growth rate and osmolar balance.

Evaluation of the Combined Administration of Chlorella fusca and Vibrio proteolyticus in Diets for Chelon labrosus: Effects on Growth, Metabolism, and Digestive Functionality.

This study aimed to evaluate the combined effect of dietary Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (C + V diet) in Chelon labrosus juveniles, highlighting their nutritional, physiological, and morphological effects. The results showed that the combined dietary inclusion of C. fusca and V. proteolyticus significantly enhanced growth performance and feed utilization compared to the control group. The C + V diet increased the fish lipid quality index (FLQ), n-3 polyunsaturated fatty acids, and n-3/n-6 ratio, which might be beneficial in terms of human nutrition. The C + V diet considerably increased carbohydrate metabolic activity by statistically boosting plasma glucose. The dietary inclusion of C. fusca in conjunction with V. proteolyticus increased metabolic enzyme activity as well as intestinal absorption capacity compared to that found in the control group. In conclusion, the experimental diet was suitable for feeding C. labrosus, increasing their growth and the nutritional characteristics of the muscle and intestine, without causing tissue damage.

Long-term assessment of the nutrient recovery capacity and biomass productivity of Scenedesmus almeriensis in raceway reactors using unprocessed urban wastewater.

The present work aims to assess the treatment of unprocessed urban wastewater using the microalga Scenedesmus almeriensis. Two 12 m3 raceway reactors, one supplemented by wastewater and the second by chemical fertilizer, operating outdoors in a semi-continuous mode, were used for eight months. Results suggested that S. almeriensis can be produced in wastewater without affecting the photosynthetic apparatus reaching a productivity of 13 g·m-2·day-1 on average in both the systems. Furthermore, the nutrient content in terms of nitrogen, phosphorous and chemical oxygen demand of the wastewater was reduced under the European limitations during most of the period, with an average removal rate of 2.2, 0.2 and 3.0 g·m-2·day-1 respectively. Therefore, raceways demonstrated a high potential for microalgal production and successful biotreatment, proving robust and reliable. Finally, the effect of environmental conditions on biomass productivity of the clean system was evaluated in a model with high accuracy (R2 = 0.9, p = 0.0002).

Rheology of microalgae concentrates and its influence on the power consumption of enzymatic hydrolysis processing.

The optimization of downstream processing is a critical step in any microalgae-related process. The microalgal biomass is separated from the initial diluted cultures to form a concentrated slurry, the properties of which greatly influence the design and performance of further processing steps, such as enzymatic hydrolysis. In this work, the rheological behaviour of two microalgal concentrates produced both in freshwater (Scenedesmus almeriensis) and seawater (Nannochloropsis gaditana) were studied. Measurements were performed on the entire range of biomass concentrations, from 0.5 g/L to 264 g/L. Non-Newtonian behaviour was observed whatever the water type and biomass concentration used, especially at high biomass concentrations above 10 g/L. The rheological data were adjusted to the Power Law model, and the consistency and flow behaviour indexes were correlated with the biomass concentration. The results show that the freshwater and seawater biomasses exhibited different behaviours, with freshwater slurries being more viscous than seawater ones. The high viscosity of freshwater slurries requires increased energy consumption for mixing, with an estimated cost increase of 60% when using them under the non-Newtonian conditions considered. These findings highlight the considerable effect of algal biomass rheology on the mixing power required during microalgal biomass processing.

Biostimulant Capacity of Chlorella and Chlamydopodium Species Produced Using Wastewater and Centrate.

The aim of the present study was to assess the potential of producing four microalgal strains using secondary-treated urban wastewater supplemented with centrate, and to evaluate the biostimulant effects of several microalgal extracts obtained using water and sonication. Four strains were studied: Chlorella vulgaris UAL-1, Chlorella sp. UAL-2, Chlorella vulgaris UAL-3, and Chlamydopodium fusiforme UAL-4. The highest biomass productivity was found for C. fusiforme, with a value of 0.38 ± 0.01 g·L-1·day-1. C. vulgaris UAL-1 achieved a biomass productivity of 0.31 ± 0.03 g·L-1·day-1 (the highest for the Chlorella genus), while the N-NH4+, N-NO3-, and P-PO43- removal capacities of this strain were 51.9 ± 2.4, 0.8 ± 0.1, and 5.7 ± 0.3 mg·L-1·day-1, respectively. C. vulgaris UAL-1 showed the greatest potential for use as a biostimulant-when used at a concentration of 0.1 g·L-1, it increased the germination index of watercress seeds by 3.5%. At concentrations of 0.5 and 2.0 g·L-1, the biomass from this microalga promoted adventitious root formation in soybean seeds by 220% and 493%, respectively. The cucumber expansion test suggested a cytokinin-like effect from C. vulgaris UAL-1; it was also the only strain that promoted the formation of chlorophylls in wheat leaves. Overall, the results of the present study suggest the potential of producing C. vulgaris UAL-1 using centrate and wastewater as well as the potential utilisation of its biomass to develop high-value biostimulants.

Biorefinery Approach Applied to the Production of Food Colourants and Biostimulants from Oscillatoria sp.

In this study, a biorefinery based on Oscillatoria sp. is developed to produce high-value compounds such as C-phycocyanin, used in food colourant applications, and biostimulants, used in agriculture-related applications. First, the Oscillatoria biomass production was optimized at a pilot scale in an open raceway reactor, with biomass productivities equivalent to 52 t/ha·year being achieved using regular fertilizers as the nutrient source. The biomass produced contained 0.5% C-phycocyanins, 95% of which were obtained after freeze-thawing and extraction at pH 6.5 and ionic strength (FI) 100 mM, with a purity ratio of 0.71 achieved in the final extract. This purity ratio allows for use of the extract directly as a food colourant. Then, the extract's colourant capacity on different beverages was evaluated. The results confirm that C-phycocyanin concentrations ranging from 22 to 106 mg/L produce colours similar to commercial products, thus avoiding the need for synthetic colourants. The colour remained stable for up to 12 days. Moreover, the safety of the extracted C-phycocyanin was confirmed through toxicity tests. The waste biomass was evaluated for use as a biostimulant, with the results confirming a relevant auxin-like positive effect. Finally, an economic analysis was conducted to evaluate different scenarios. The results confirm that the production of both C-phycocyanin and biostimulants is the best scenario from an economic standpoint. Therefore, the developed biomass processing scheme provides an opportunity to expand the range of commercial applications for microalgae-related processes.

Optimisation of Operational Conditions during the Production of Arthrospira platensis Using Pilot-Scale Raceway Reactors, Protein Extraction, and Assessment of their Techno-Functional Properties.

The aim of the present study was to identify the optimum combination of dilution rate and depth of the culture to maximise the Arthrospira platensis BEA005B (Spirulina) productivity using 80 m2 raceway reactors. By varying these two main operational conditions, the areal biomass productivity of the reactors varied by over 55%. The optimum combination, optimised using a surface response methodology, was a depth of 0.10 m and a dilution rate of 0.33 day-1, which led to a biomass productivity of 30.2 g·m-2·day-1 on a dry weight basis when operating the reactors in semi-continuous mode. The composition of the produced biomass was 62.2% proteins, 42.5% carbohydrates, 11.6% ashes, and 8.1% lipids. The isolated proteins contained all the essential amino acids (except for tryptophan, which was not determined); highlighting the content of valine (6.8%), histidine (8.3%), and lysine (7.5%). The functional properties of the proteins were also assessed, demonstrating huge potential for their use in the development of innovative and sustainable foods.

Towards large scale biocrust restoration: Producing an efficient and low-cost inoculum of N-fixing cyanobacteria.

Dryland soil degradation is increasing due to global change and traditional restoration methods are not successful due to water scarcity. Thus, an alternative technology based on inoculating biocrust-forming cyanobacteria on degraded soils has emerged. Biocrusts are communities of mosses, lichens, cyanobacteria or fungi that colonize soil surface forming a stable and fertile layer. Previous studies have shown the benefits of inoculating cyanobacteria to restore soils at a small scale. However, to face field restoration projects, it is necessary to produce high quantities of biomass at an affordable cost. In this work, we analyze if the previously tested cyanobacteria Scytonema hyalinum, Tolypothrix distorta (heterocystous strains) and Trichocoleus desertorum (a bundle-forming one) can be produced with agricultural fertilizers. Different culture media were used: two containing pure chemicals (BG11 and BG110, this N-free medium was used just for heterocystous strains) and two containing fertilizers (BG11-F and MM-F). The performance of the cultures was monitored by measuring the biomass concentration and photosynthetic stress. Afterwards, we analyzed their capacity to induce biocrusts and improve soil properties by inoculating the biomass on a mine substrate indoors and measuring, three months later, the albedo, chlorophyll a and organic carbon content. Results show that the bundle-forming cyanobacterium was unable to grow in the media tested, whereas both heterocystous cyanobacteria grew in all of them and induced the formation of biocrusts improving the organic carbon substrate content. The best results for S. hyalinum were found using the MM-F medium, and for T. distorta using a medium containing pure chemicals (BG11). However, results were also positive when using a medium containing fertilizers (BG11-F). Thus, agricultural fertilizers can be used to undertake the production of heterocystous cyanobacteria for large scale restoration in drylands. On the other hand, more research is needed to find sustainable techniques to produce biomass of bundle-forming cyanobacteria.

A seasonal simulation approach for culture depth influence on the temperature for different characterized microalgae strains.

Irradiance and temperature are among the most important variables that affect microalgae growth, being both difficult to control in outdoor raceway reactors utilized for large-scale production of microalgae biomass. They are mainly a function of the location of the reactors, thus, producing certain strains of microalgae in inappropriate places conduces to the failure of the systems. To be able to determine important parameters of any microalgae strains on the performance of the culture, such as the influence of irradiance and temperature, is a powerful tool in decision-making processes. In addition, whatever the strain and location, operation strategies must be defined for each specific case, such as the imposed dilution rate and culture depth, both influencing the light availability and temperature of the culture as major variables determining the biomass productivity. In this paper, a simulation-based methodology is proposed to establish the influence of season and culture depth on the 1-year age irradiance and temperature of the culture, and thus on the biomass productivity of different microalgae strains. Up to five of the most frequently produced strains, such as Spirulina platensis, Chlorella vulgaris, Nannochloropsis gaditana, Isochrysis galbana, and Scenedesmus almeriensis have been considered. The challenge is to develop an easy-to-manage decision-making tool for the optimal design and operation of large-scale microalgae facilities. Especially, dates for microalgae production and culture depth at which the reactors must be operated will be provided, being valid for any microalgae strain. The proposed methodology will largely contribute to the risk of investment in this field, then to enlarge the relevance of the microalgae production industry.

Microalgae for the Food Industry: From Biomass Production to the Development of Functional Foods.

The human population is expected to reach 9 [...].

Annual assessment of the wastewater treatment capacity of the microalga Scenedesmus almeriensis and optimisation of operational conditions.

The depth of the culture and the dilution rate have a striking effect on the biomass productivity and the nutrient recovery capacity of microalgal cultures. The combination of culture depth and dilution rate that allows to maximise the performance of the system depends on environmental conditions. In the current study, a response surface methodology was used to explore the relationship between the two most relevant operational conditions and the biomass productivity achieved in 8.3 m2 pilot-scale raceways operated using urban wastewater. Four polynomial models were developed, one for each season of the year. The software predicted biomass productivities of 12.3, 25.6, 32.7, and 18.9 g·m-2·day-1 in winter, spring, summer, and autumn, respectively. The models were further validated at pilot-scale with R2 values ranging within 0.81 and 0.91, depending on the season. Lower culture depths had the advantage of minimising nitrification and stripping but allow to process a lower volume of wastewater per surface area. Biomass productivity was higher at culture depths of 0.05 m, when compared to 0.12 and 0.20 m, while the optimal dilution rate was season-dependent. Results reported herein are useful for optimising the biomass productivity of raceway reactors located outdoors throughout the year.

Evaluation of the dynamics of microalgae population structure and process performance during piggery wastewater treatment in algal-bacterial photobioreactors.

The dynamics of microalgae population during piggery wastewater (PWW) treatment in four open photobioreactors operated at 27days of hydraulic retention time, and inoculated with Chlorella sp. (R1), Acutodesmus obliquus (R2), Oscillatoria sp. (R3) and in the absence of inoculum (R4), were evaluated for 6months. In addition, the algal-bacterial biomass concentration, removal of organic matter, nutrients and heavy metals were also assessed. The results revealed a high diversity and rapid variations in the structure of microalgae populations, Chlorella sp. being dominant in R4 throughout most of the operational period. Steady state average biomass concentration ranged from 2445-2610mg/L in R1-R3 to 3265mg/L in R4. No significant differences were recorded in the removal efficiencies (REs) of total organic carbon (86-87%), inorganic carbon (62-71%), total nitrogen (82-85%) and total phosphorous (90-92%). Finally, Zn-REs accounted for 26% in R3, 37% in R2, and 49% in R1 and R4.

Comparative evaluation of piggery wastewater treatment in algal-bacterial photobioreactors under indoor and outdoor conditions.

This work evaluated the performance of four open algal-bacterial photobioreactors operated at ≈26days of hydraulic retention time during the treatment of 10 (×10) and 20 (×20) times diluted piggery wastewater (PWW) under indoor (I) and outdoor (O) conditions for four months. The removal efficiencies (REs) of organic matter, nutrients and zinc from PWW, along with the dynamics of biomass concentration and structure of algal-bacterial population were assessed. The highest TOC-RE, TP-RE and Zn-RE (94±1%, 100% and 83±2%, respectively) were achieved indoors in ×10 PWW, while the highest TN-RE (72±8%) was recorded outdoors in ×10 PWW. Chlorella vulgaris was the dominant species regardless of the ambient conditions and PWW dilution. Finally, DGGE-sequencing of the bacterial community revealed the occurrence of four phyla, Proteobacteria being the dominant phylum with 15 out of the 23 most intense bands.