Helena M. Amaro

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.

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.

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.

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 β‐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 β‐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.

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.

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.

Applications of Spent Biomass

Abstract There are several difficulties in developing algal biofuels that can fully replace fossil fuels; the main challenges relate to lower environmental impact (including the possibility of CO2 sequestering and wastewater treatment) and economic feasibility. To date, most studies have focused on selection of species, coupled with optimization of cultivation, biomass harvest and fuel extraction; efforts on enhancing environmental benefits and upgrading spent biomass are thus in order. Industry should indeed adopt an integrated strategy inspired by algal-based biorefineries – toward maximizing the economic return of all products present in algal biomass, by recovering high value compounds prior to biofuel manufacture, and using the remainder for aquaculture/land animal feed and production of other biofuels via supplementary fermentation.

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 β-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.

Microalgal fatty acids—From harvesting until extraction

Abstract Microalgal biomass can be “energy rich,” but the growth of algae in dilute suspension around 0.02%–0.05% dry solids is a considerable challenge in achieving a viable energy balance in a microalgal biofuel process. Many researchers consider that efficient harvesting is the major challenge of microalgal biofuel commercialization but a single method or combination of harvesting methods suited to all microalgae does not exist. Additionally, lipid extraction continues to be a significant trial even though a multitude of extraction methods, especially in what concerns the presence of residual water in the microalgal biomass; its effects on lipid extraction or the need of cell disruption are still not well understood. In this chapter, these critical points on biofuel production—biomass harvesting and dewatering, biomass pretreatment, and fatty acid extraction methods—will be discussed.