Microalgae systems - environmental agents for wastewater treatment and further potential biomass valorisation.

Water is the most valuable resource on the planet. However, massive anthropogenic activities generate threatening levels of biological, organic, and inorganic pollutants that are not efficiently removed in conventional wastewater treatment systems. High levels of conventional pollutants (carbon, nitrogen, and phosphorus), emerging chemical contaminants such as antibiotics, and pathogens (namely antibiotic-resistant ones and related genes) jeopardize ecosystems and human health. Conventional wastewater treatment systems entail several environmental issues: (i) high energy consumption; (ii) high CO2 emissions; and (iii) the use of chemicals or the generation of harmful by-products. Hence, the use of microalgal systems (entailing one or several microalgae species, and in consortium with bacteria) as environmental agents towards wastewater treatment has been seen as an environmentally friendly solution to remove conventional pollutants, antibiotics, coliforms and antibiotic resistance genes. In recent years, several authors have evaluated the use of microalgal systems for the treatment of different types of wastewater, such as agricultural, municipal, and industrial. Generally, microalgal systems can provide high removal efficiencies of: (i) conventional pollutants, up to 99%, 99%, and 90% of total nitrogen, total phosphorus, and/or organic carbon, respectively, through uptake mechanisms, and (ii) antibiotics frequently found in wastewaters, such as sulfamethoxazole, ciprofloxacin, trimethoprim and azithromycin at 86%, 65%, 42% and 93%, respectively, through the most desirable microalgal mechanism, biodegradation. Although pathogens removal by microalgal species is complex and very strain-specific, it is also possible to attain total coliform and Escherichia coli removal of 99.4% and 98.6%, respectively. However, microalgal systems' effectiveness strongly relies on biotic and abiotic conditions, thus the selection of operational conditions is critical. While the combination of selected species (microalgae and bacteria), ratios and inoculum concentration allow the efficient removal of conventional pollutants and generation of high amounts of biomass (that can be further converted into valuable products such as biofuels and biofertilisers), abiotic factors such as pH, hydraulic retention time, light intensity and CO2/O2 supply also have a crucial role in conventional pollutants and antibiotics removal, and wastewater disinfection. However, some rationale must be considered according to the purpose. While alkaline pH induces the hydrolysis of some antibiotics and the removal of faecal coliforms, it also decreases phosphates solubility and induces the formation of ammonium from ammonia. Also, while CO2 supply increases the removal of E. coli and Pseudomonas aeruginosa, as well as the microalgal growth (and thus the conventional pollutants uptake), it decreases Enterococcus faecalis removal. Therefore, this review aims to provide a critical review of recent studies towards the application of microalgal systems for the efficient removal of conventional pollutants, antibiotics, and pathogens; discussing the feasibility, highlighting the advantages and challenges of the implementation of such process, and presenting current case-studies of different applications of microalgal systems.

Karlodinium veneficum: Growth optimization, metabolite characterization and biotechnological potential survey.

AIM OF THIS STUDY: The major aim of this work was to consistently optimize the production of biomass of the dinoflagellate Karlodinium veneficum and evaluate its extracts biotechnological potential application towards food, nutraceutical or/and pharmaceutical industries. METHODS AND RESULTS: A successful approach of biomass production of K. veneficum CCMP 2936 was optimized along with the chemical characterization of its metabolite profile. Several temperatures (12, 16, 20, 25, 30°C), L1 nutrient concentrations (0.5×, 2×, 2.5×, 3×) and NaCl concentrations (20, 25, 30, 40 g L-1 ) were tested. The growth rate was maximum at 16°C, 2× nutrient concentration and 40 g L-1 of NaCl; hence, these conditions were chosen for bulk production of biomass. Methanolic extracts were prepared, and pigments, lipids and phenolic compounds were assessed; complemented by antioxidant and anti-inflammatory capacities, and cytotoxicity. Fucoxanthin and derivatives accounted for 0.06% of dry weight, and up to 60% (w/w) of all quantified metabolites were lipids. Said extracts displayed high antioxidant capacity, as towards assessed via the NO•- and ABTS•+ assays (IC50  = 109.09 ± 6.73 and 266.46 ± 2.25 µgE  ml-1 , respectively), unlike observed via the O2 •- assay (IC25 reaching 56.06 ± 5.56 µgE  ml-1 ). No signs of cytotoxicity were observed. CONCLUSIONS: Karlodinium veneficum biomass production was consistently optimized in terms of temperature, L1 nutrient concentrations and NaCl concentration. In addition, this strain appears promising for eventual biotechnological exploitation. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides fundamental insights about the growth and potential of value-added compounds of dinoflagellate K. veneficum. Dinoflagellates, as K. veneficum are poorly studied regarding its biomass production and added-value compounds for potential biotechnological exploitation. These organisms are difficult to maintain and grow in the laboratory. Thus, any fundamental contribution is relevant to share with the scientific community.

Gloeothece sp.-Exploiting a New Source of Antioxidant, Anti-Inflammatory, and Antitumor Agents.

Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from Gloeothece sp.-using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant (ABTS•+, DPPH•, •NO and O2•assays), anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity (death by TUNEL, and anti-proliferative by BrdU incorporation assay in AGS cancer cells); while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge -NO• radical (IC50, 1258 ± 0.353 µg·mL-1); (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL-1; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL-1. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.

Gloeothece sp. as a Nutraceutical Source-An Improved Method of Extraction of Carotenoids and Fatty Acids.

The nutraceutical potential of microalgae boomed with the exploitation of new species and sustainable extraction systems of bioactive compounds. Thus, a laboratory-made continuous pressurized solvent extraction system (CPSE) was built to optimize the extraction of antioxidant compounds, such as carotenoids and PUFA, from a scarcely studied prokaryotic microalga, Gloeothece sp. Following "green chemical principles" and using a GRAS solvent (ethanol), biomass amount, solvent flow-rate/pressure, temperature and solvent volume-including solvent recirculation-were sequentially optimized, with the carotenoids and PUFA content and antioxidant capacity being the objective functions. Gloeothece sp. bioactive compounds were best extracted at 60 °C and 180 bar. Recirculation of solvent in several cycles (C) led to an 11-fold extraction increase of ß-carotene (3C) and 7.4-fold extraction of C18:2 n6 t (5C) when compared to operation in open systems. To fully validate results CPSE, this system was compared to a conventional extraction method, ultrasound assisted extraction (UAE). CPSE proved superior in extraction yield, increasing total carotenoids extraction up 3-fold and total PUFA extraction by ca. 1.5-fold, with particular extraction increase of 18:3 n3 by 9.6-fold. Thus, CPSE proved to be an efficient and greener extraction method to obtain bioactive extract from Gloeothece sp. for nutraceutical purposes-with low levels of resources spent, while lowering costs of production and environmental impacts.

Effect of Solvent System on Extractability of Lipidic Components of Scenedesmus obliquus (M2-1) and Gloeothece sp. on Antioxidant Scavenging Capacity Thereof.

Microalgae are well known for their biotechnological potential, namely with regard to bioactive lipidic components-especially carotenoids and polyunsaturated fatty acids (PUFA), well-known for therapeutic applications based on their antioxidant capacity. The aim of this work was to evaluate the influence of four distinct food-grade solvents upon extractability of specific lipidic components, and on the antioxidant capacity exhibited against both synthetic (2,2-diphenyl-1-picrylhydrazyl (DPPH(•)) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+•))) and biological reactive species (O2(•)⁻ and (•)NO⁻). A eukaryotic microalga (Scenedesmus obliquus (M2-1)) and a prokaryotic one (Gloeothece sp.) were used as case studies. Concerning total antioxidant capacity, the hexane:isopropanol (3:2) and acetone extracts of Sc. obliquus (M2-1) were the most effective against DPPH(•) and ABTS(+•), respectively. Gloeothece sp. ethanol extracts were the most interesting scavengers of O2(•)⁻, probably due the high content of linolenic acid. On the other hand, acetone and hexane:isopropanol (3:2) extracts were the most interesting ones in (•)NO⁻ assay. Acetone extract exhibited the best results for the ABTS assay, likely associated to its content of carotenoids, in both microalgae. Otherwise, ethanol stood out in PUFA extraction. Therefore, profiles of lipidic components extracted are critical for evaluating the antioxidant performance-which appears to hinge, in particular, on the balance between carotenoids and PUFAs.

Effects of Temperature, pH, and NaCl Concentration on Biomass and Bioactive Compound Production by Synechocystis salina.

Synechocystis salina is a cyanobacterium that has biotechnological potential thanks to its ability to synthesize several bioactive compounds of interest. Therefore, this study aimed to find optimal conditions, in terms of temperature (15-25 °C), pH (6.5-9.5), and NaCl concentration (10-40 g·L-1), using as objective functions the productivities of biomass, total carotenoids, total PBPs, phycocyanin (PC), allophycocyanin (APC), phycoerythrin (PE), and antioxidants (AOXs) capacity of Synechocystis salina (S. salina) strain LEGE 06155, based in factorial design resorting to Box-Behnken. The model predicted higher biomass productivities under a temperature of 25 °C, a pH of 7.5, and low NaCl concentrations (10 g·L-1). Maximum productivities in terms of bioactive compounds were attained at lower NaCl concentrations (10 g·L-1) (except for PE), with the best temperature and pH in terms of carotenoids and total and individual PBPs ranging from 23-25 °C to 7.5-9.5, respectively. PE was the only pigment for which the best productivity was reached at a lower temperature (15 °C) and pH (6.5) and a higher concentration of NaCl (≈25 g·L-1). AOX productivities, determined in both ethanolic and aqueous extracts, were positively influenced by lower temperatures (15-19 °C) and higher salinities (≈15-25 g·L-1). However, ethanolic AOXs were better recovered at a higher pH (pH ≈ 9.5), while aqueous AOXs were favored by a pH of 8. The model showed that biomass production can be enhanced by 175% (compared to non-optimized conditions), total carotenoids by 91%, PC by 13%, APC by 50%, PE by 130%, and total PBPs by 39%; for AOX productivities, only water extracts exhibited a (marginal) improvement of 1.4%. This study provided insightful information for the eventual upgrading of Synechocystis salina biomass in the biotechnological market.

Factorial Optimization of Ultrasound-Assisted Extraction of Phycocyanin from Synechocystis salina: Towards a Biorefinery Approach.

PC is a bioactive and colorant compound widely sought in the food, nutraceutical and cosmetic industries, and one of the most important pigments produced by Synechocystis salina. However, the general extraction process is usually time-consuming and expensive, with low extraction yields-thus compromising a feasible and sustainable bioprocess. Hence, new extraction technologies (e.g., ultrasound assisted-extraction or UAE) emerged in the latest years may serve as a key step to make the overall bioprocess more competitive. Therefore, this study aimed at optimizing the yields of phycocyanin (PC) rich-extracts of S. salina by resorting to UAE; in attempts to explore this process in a more economically feasible way; valorization of the remaining cyanobacterial biomass, via extraction of other bioactive pigments and antioxidants, was tackled within a biorefinery perspective. A two-stage extraction (using ethanol and water) was thus performed (because it favors PC extraction); other bioactive pigments, including chlorophyll a (chl a), carotenoids, and other phycobiliproteins (PBPs), but also antioxidant (AOX) capacity and extraction yields were also evaluated for their optimum UAE yields. A factorial design based on Box-Behnken model was developed; and the influence of such extraction parameters as biomass to solvent ratio (B/S ratio = 1.5-8.5 mg·mL-1), duty cycle (DT = 40-100%), and percentage of amplitude (A = 40-100%) were evaluated. The model predicted higher PC yields with high B/S ratio = 6 mg·mL-1, lower DT = 80% and an A = 100%. Classical extraction was compared with UAE under the optimum conditions found; the latter improved PC yields by 12.5% and 47.8%, when compared to freeze-thawing extraction, and bead beater homogenization-based extraction, respectively. UAE successive extractions allowed to valorize other important bioactive compounds than PC, by reusing biomass, supporting a favorable contribution to the economic feasibility of the S. salina-based process towards a biorefinery approach.

Potential of Microalgae Extracts for Food and Feed Supplementation-A Promising Source of Antioxidant and Anti-Inflammatory Compounds.

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

Microalgae as sources of carotenoids.

Marine microalgae constitute a natural source of a variety of drugs for pharmaceutical, food and cosmetic applications-which encompass carotenoids, among others. A growing body of experimental evidence has confirmed that these compounds can play important roles in prevention (and even treatment) of human diseases and health conditions, e.g., cancer, cardiovascular problems, atherosclerosis, rheumatoid arthritis, muscular dystrophy, cataracts and some neurological disorders. The underlying features that may account for such favorable biological activities are their intrinsic antioxidant, anti-inflammatory and antitumoral features. In this invited review, the most important issues regarding synthesis of carotenoids by microalgae are described and discussed-from both physiological and processing points of view. Current gaps of knowledge, as well as technological opportunities in the near future relating to this growing field of interest, are also put forward in a critical manner.

Continuous pressurized extraction versus electric fields-assisted extraction of cyanobacterial pigments.

Cyanobacteria pigments, in special carotenoids and phycobiliproteins, are usually used in industry as raw extracts, although there is still no standard methodology for their extraction. For the co-extraction of carotenoids and phycobiliproteins from the marine cyanobacterium Cyanobium sp., a continuous pressurized solvent extraction (CPSE) system and an electric fields-assisted extraction system based in ohmic heating were optimized using Central Composite Designs, with three factors each: time (t), temperature (T) and, flow (f) for CPSE; and time, temperature and frequency (F) for ohmic heating. The content of pigments and the antioxidant capacity of extracts were evaluated. All tested factors seem to influence the extraction of pigments in different ways: a high temperature (70 °C) had a positive impact on the extraction rate in both methods, while the influence of time depended on the extraction principle. Flow and frequency affected directly the extraction efficiency and these methods are indeed suitable for cyanobacterial pigments extraction, achieving good extraction results. Optimal conditions for co-extraction of carotenoids and phycobiliproteins in CPSE were T = 70 °C, t = 20 min and f = 1.5 mL min-1, and for ohmic heating they were T = 70 °C, t = 5 min and F = 20 kHz. Both, CPSE and ohmic heating systems allowed obtaining better extraction yields when compared with a previously optimized extraction method (homogenization), used here as a reference. However, ohmic heating was the best methodology for pigments co-extraction from Cyanobium sp.

Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications.

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds.

Microalgal compounds modulate carcinogenesis in the gastrointestinal tract.

Gastrointestinal cancers rank second in overall cancer-related deaths. Carotenoids, sulfated polysaccharides, and polyunsaturated fatty acids (PUFAs) from microalgae exhibit cancer chemopreventive features at different stages of carcinogenesis. For instance, sulfated polysaccharides bear a prophylactic potential via blocking adhesion of pathogens to the gastric surface, whereas carotenoids are effective against Helicobacter pylori infection. This effect is notable because H. pylori has been targeted as the primary cause of gastric cancer. Recent results on antitumor and antibacterial compounds synthesized by microalgae are reviewed here, with an emphasis on their impact upon H. pylori infection and derived pathologies accompanying the progression of gastric carcinogenesis.

Optimization of ABTS radical cation assay specifically for determination of antioxidant capacity of intracellular extracts of microalgae and cyanobacteria.

A renewed interest in antioxidants has arisen in recent years; microalgae and cyanobacteria are potential sources thereof for use as food/feed ingredients. However, improved methods for comprehensive screening of antioxidant capacity specifically in intracellular extracts of marine microorganisms are required - encompassing lipophilic and hydrophilic compounds simultaneously. The original ABTS method was thus improved, and in particular the procedures of cell disruption and storage were optimized. The best solvent found was ethanol/water (1:1, v/v). The reaction to form ABTS(+) in said solvent was essentially complete by eight hours, and this radical cation was stable for at least 6 days; at room temperature, the ABTS(+) solution remained within an allowable analytical range for up to 13 h. Ultra Turrax was the best cell disruption method, and refrigeration was the best preservation method. This improved methodology was validated with four representative strains that respond poorly to cell disruption.

Microalgae as sources of high added-value compounds--a brief review of recent work.

Microalgae have found commercial applications as natural sources of valuable macromolecules, including carotenoids, long-chain polyunsaturated fatty acids, and phycocolloids. As photoautotrophs, their simple growth requirements make them attractive for bioprocesses aimed at producing high added-value compounds that are in large demand by the pharmaceutical market. A few compounds synthesized by microalgae have indeed proven to possess anti-inflammatory, antiviral, antimicrobial, and antitumoral features; astaxanthin, a known antioxidant produced by Haematococcus pluvialis, is an illustrative example with important anti-inflammatory and antitumoral roles. From a chemical standpoint, several such compounds are polysaccharides or long chain fatty acids, where the latter can be either saturated or unsaturated. Additionally, their chemical structures are often atypical, whereas their concentrations can exceed those found in many other natural sources. The productivity and biochemical composition of microalgae depend strongly on the mode of cultivation, medium composition, and nutrient profile. Consequently, numerous efforts aimed at elucidating the practical impacts of the aforementioned parameters have been developed. This review accordingly covers the knowledge produced in the last two decades on the uses of microalgae to obtain physiologically active compounds, and on the optimization of the underlying production and purification processes. It also identifies major gaps and opportunities in this field that should be addressed or exploited in the near future.

Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus.

Reactive forms of oxygen can damage DNA (among other molecules), thus triggering, e.g., atherogenesis and carcinogenesis. However, such dietary antioxidants as lutein and ß-carotene can effectively inactivate them; these compounds were found to high levels in a novel strain (M2-1) of the microalga Scenedesmus obliquus. The independent and combined effects of pH and temperature on its rates of growth and production of antioxidants were experimentally assessed, via a full factorial experimental design; the effects of each parameter independently, and of their interactions were accordingly quantified by ANOVA. Our results indicated that temperature plays a more important role on the maximum specific growth rate than pH; in terms of antioxidant content, pH and, to a lesser extent, temperature also have relevant effects. Consequently, the highest rate of biomass specific growth (0.294 ± 0.013 day(-1)) and biomass productivity (0.837 ± 0.054 mg L(-1) day(-1)) were associated with relatively low pH (6) and relatively high temperature (30°C). Conversely, the antioxidant production rate increased with pH; hence, the highest productivity (0.638 mg L(-1) day(-1)) was attained at pH 8 and 30°C. At the best operating conditions for antioxidant content, the levels of lutein and ß-carotene were 203.57 ± 1.41 and 18.20 ± 0.33 mg mL(-1), respectively; the maximum production of either one occurred at the early exponential phase.

Efficient H2 production via Chlamydomonas reinhardtii.

Molecular hydrogen (H(2)) obtained from biological sources provides an alternative to bulk chemical processes that is moving towards large-scale, economical generation of clean fuel for automotive engines. This opinion article examines recent improvements in H(2) production by wild and mutant strains of Chlamydomonas reinhardtii - the green microalga currently considered the best eukaryotic H(2) producer. Here, we review various aspects of genetic and metabolic engineering of C. reinhardtii, as well as of process engineering. Additionally, we lay out possible scenarios that would lead to more efficient research approaches in the near future, as part of a consistent strategy for sustainable biohydrogen supply.