Marine versatility

Seaweed may be used for cleaning up contaminated areas

Eduardo CesarGrowing Gracilaria tenuistipitata in the laboratoryEduardo Cesar

With their different shapes and colors delicate marine algae contain an extremely rich chemical arsenal, composed of amino acids, lipids, sugars, carotenoids and pigments that make them particularly interesting as a source of new drugs and bioactive substances that have economic potential for use in agriculture, or even for producing biofuel. Versatile, these aquatic organisms can also be used for cleaning areas contaminated by organic substances and heavy metals, a process called bioremediation. “In their cell structure algae have a large area called the vacuole, a kind of cavity surrounded by a membrane, where they manage to store large quantities of chemicals”, says Professor Pio Colepicolo Neto, from the Department of Biochemistry at the Institute of Chemistry of the University of São Paulo (USP), who for over 20 years has dedicated himself to the study of algae and is currently coordinating a thematic project involving nine research groups, which is funded by FAPESP and covers studies for bioprospecting marine macroalgae. “In an area with heavy metals they can function as a biological sponge, absorbing these pollutants, and using biochemical mechanisms, inside the cell the material is immobilized in the vacuole,” he reports. At the end of the process, they only have to be incinerated to remove the metal that is concentrated in the ashes. To test the knowledge of years of research in practice, Colepicolo recently delivered a project to Petrobras, which is being analyzed by the company, to use macroalgae in refinery tanks for cleaning up the heavy metals resulting from oil production processes.

One of the proposals included in the project is to study the concentration levels of carbon dioxide (CO2), not only in refineries, but also in ethanol fermentation plants so that this atmospheric pollutant can be piped and pumped for growing seaweed. “With the absorption of carbon dioxide it will be possible to help clean up the atmosphere and as a result earn carbon credits,” says Colepicolo. Seaweed is at the base of the food chain and generates important biomolecules, such as antioxidants, essential amino acids, vitamins, carotenoids, polysaccharides and fatty acids, like omega-3 and omega-6. “The carbon dioxide will act as food for the algae to gain biomass,” says the researcher.

In studies conducted in partnership with the Center for Training and Research in the Environment (CEPEMA), which is linked to USP and headquartered in Cubatão in the lower Santos Basin region, the research group from the Institute of Chemistry has tested the degradation of some organic pollutants, such as phenol, by seaweed. In addition to being able to degrade an extremely toxic compound, the algae use the carbon from phenol to build amino acids, lipids and nucleic acids. “The chemical structures of different compounds from seaweed are completely different from the structures produced by terrestrial plants,” says Colepicolo. As they live in a highly adversarial environment, where they are attacked all the time by other organisms that feed on them and use them as shelter, they have a diversified range of extremely sophisticated chemicals to defend themselves. One of these substances is microsporine-like amino acids (MMAs), a chemical compound with a low molecular weight that is synthesized by algae and fungi that have a high capacity for absorbing ultraviolet radiation and that were isolated and characterized in USP’s laboratory . “We isolated more than 20 microsporine from different macroalgae of the Gracilaria family, found on the Brazilian coast. Early in the project our objective was to develop an approach only involving basic science, but with their high capacity for absorbing ultraviolet (UV) light, it was inevitable to think of the application of these molecules in many different products that are exposed to sunlight. In addition to sunscreens, these substances can be used directly on material or in paints and varnishes for homes and boats,” says Colepicolo.

Jean-Paul SorianoDetail of fronds of Caulerpa racemosaJean-Paul Soriano

One of the extracts obtained proved to have excellent potential for use in cosmetic formulations intended for sun protection. The project to obtain a natural photoprotective substance was developed in partnership with Natura, as part of the Partnership for Technological Innovation (Pite) program, financed by FAPESP. “The amount of ultraviolet radiation they absorb is extremely high and comparable with the synthetic compounds used in the composition of current commercial sunscreens,” says Colepicolo. “A big difference is that these micosporines absorb UVB (in the spectral region from 280 to 330 nanometers), where few molecules do.” With the gradual increase of sunlight on the planet, there is a need for protection in the UVB region. “The addition of natural substances with the same efficiency as synthetic ones adds value to the product, making it special and with a competitive market value,” says the researcher. Natura has already carried out stability tests on the substance and an assessment of its cytotoxicity, a trial made on cultured cells, which is necessary for checking the biocompatibility of the materials. “In the cytotoxicity trials it was seen that microsporine does not kill cells, either by absorption of ultraviolet radiation or from the effect of white light.”

Besides microsporines, seaweed produces several other compounds with anti-inflammatory, bactericide and fungicide properties.” Some substances extracted from seaweed, when sprayed on papaya, figs and eggplants, increase the shelf life of these products,” says Colepicolo. “In some vegetables there was an increase of 30 days in the shelf life after application.” However, until it is considered a product it is necessary to test the toxicity of substances post-consumption and also the effects they may have on the fruit to which they are applied; this work is likely to take about two years. To get to substances of interest, the researchers analyzed dozens of species of algae. “After grinding the seaweed, we prepared extracts with different chemical polarities and we tested them on a large scale,” says Colepicolo. In the United States, some producers are using seaweed extracts enriched with carotenoids mixed with the food for laying hens to give a more attractive color to the eggs, and make the birds healthier, into the bargain. This is because the carotenoids found in these organisms are precursors of the synthesis of vitamin A in animals. The main problem with widespread use is that these substances are still expensive. “A dozen eggs with seaweed extracts can cost between $4 and $5,” says Colepicolo.

Integrated farming
One of the lines of bioremediation being considered focuses on the integrated farming of shrimp and algae. In this case, the macroalgae are grown in nurseries similar to tanks on the farms that produce shellfish in Rio Grande do Norte. Studies have shown that the wastewater from intensive aquaculture, which is rich in nitrogen and phosphorus, can be used as a source of nutrients for growing macroalgae. In this case, it is important to find a species of alga that is tolerant or resistant to a particular nutrient in order to improve production potential and bioremediation. As a result of this partnership, the environment becomes more balanced and favorable to the growth of cultivated organisms. Two farms are participating in the project, coordinated by Professor Eliane Marinho-Soriano, from the Department of Oceanography and Limnology, at the Federal University of Rio Grande do Norte (UFRN). One of them is Primar, the only certified organic shrimp farming company in Brazil, and the other is Tecnarão, which is owned by an Argentinean group. Both are located on the banks of the Guaraíras Lagoon, 70 km from Natal, bordered by mangrove swamps. In nurseries about 1.5 meters deep and covering 3 to 4 hectares each, the shrimp are fed with vitamin-enhanced feed several times a day until they reach commercial size. At the end of three months, when the shrimp are ready to be collected, the water used in the cultivation is returned to the natural environment, resulting in an excessive increase in the nutrient load. When algae (Gracilaria domingensis and Gracilaria birdiae) are cultivated in shrimp ponds, they feed on the detritus expelled by the shellfish, which ultimately leads to cleaner water that can be returned to the mangrove swamps or reused in cropping systems. The biomass from the macroalgae produced in these systems can be used for human food, pet food and bioactive compounds with high economic value.

Eliane is also coordinating a project for growing algae in the open sea on Rio do Fogo beach, 80 km from Natal, an activity being carried out in a partnership between the UFRN, the Ministry of Fishing and Agriculture and fishing families from the community. The work began in 2001 as a pilot project, funded by the United Nations Food and Agriculture Organization (FAO) to help in the development of needy communities. “We showed that it was economically viable to grow algae in this region,” says Eliane. The project encompasses the management of seaweed banks, the restoration of these banks, technical assistance and the supply of data to environmental bodies. Currently, about 25 people are involved in the collection of Gracilaria, a species native to the northeastern coast. In Rio do Fogo the cultivation of Gracilaria was set up in 2005, approximately 50 meters from the beach in cropping systems called floating rafts. These structures are made from PVC pipes and ropes, which stay on the surface of the water with the help of buoys. The stems of the algae (cuttings) are attached to the ropes. After three months, the seaweed is ready to be harvested. Like land plants, the macroalgae carry out photosynthesis and are capable of converting solar energy into chemical energy by metabolizing compounds in materials needed for their growth. In addition to chlorophyll, which is responsible for the basic photosynthesis process, the algae have other pigments that give them different colors, ranging from light green to purple. Based on the pigmentation, the macroalgae are classified into the green (Chlorophyta), brown (Ochrophyta) or red (Rhodophyta) algae groups, to which the Gracilaria species belong.

Jean-Paul SorianoNatural clumps of GracilariaJean-Paul Soriano

Substantial value
“Depending on the species, seaweed can be found both in shallower areas as well as at greater depths,” says Eliane, who in 2009 published the “Manual for the identification of marine macroalgae on the coast of Rio Grande do Norte”, a simple and practical field guide. Because of its nutritional and medicinal properties, seaweed has long been used by people from the East. However, it is some of the polysaccharides, such as agar and carrageenan, used in the food, pharmaceutical and cosmetic industries as stabilizers, softening agents and thickeners, which give these aquatic organisms substantial economic value. “Around 25,000 tons of carrageenan are consumed every year, which corresponds to $200 million,” says Colepicolo. It is a market that is growing by 5 percent a year. Agar is responsible for 10,000 tons annually, or $10 million, and is growing by 7 percent per annum. “All the carrageenan used in Brazil is still imported because we have do not have the production to meet domestic market needs,” he emphasizes. In Brazil, until a few years ago, there were several companies that processed seaweed, but it was not cultivated; it was extracted from nature, which resulted in a shortage of raw material. Today only one company in Paraíba processes seaweed. “To keep companies running, there needs to be biomass available and the only way of doing this is by growing it, as they do in Chile, Indonesia, Japan and China,” says Eliane.

One line of research in the thematic project deals with the cultivation and production of seedlings in the laboratory, a task being carried out by researchers Nair Sumie Yokoya and Mutue Toyota Fujii, from the Botanical Institute, which is linked to the São Paulo State Department of the Environment. This part of the project generates seedlings of macroalgae that can be grown at different temperatures and salinities. “The research undertaken at the Botanical Institute is fundamental to the project’s success, because in a country where the coast is 8000 km long and has different climatic conditions, it is necessary to distribute the seedlings according to their tolerance and capacity to grow and produce bioactive varieties,” says Colepicolo. Nair is also the coordinator of the National Network in Marine Macroalgae Biotechnology, which was set up in 2005. Also participating in the project is Professor Ernani Pinto, from USP’s School of Pharmaceutical Sciences, which is conducting research into the production of bioactive seaweed and coordinating the project’s pharmacological trials, as well as an analysis of bioprospecting, feasibility and market studies. “Because of its complexity and diversity, molecules of macroalgae can fill important gaps in the discovery of new drugs,” says Ernani. Professor Norbert Peporine Lopes” group, from USP’s School of Pharmaceutical Sciences in Ribeirao Preto, is responsible for explaining the structure of the chemical substances isolated from macroalgae with bioactivity, and a group of researchers from the Federal University of Santa Catarina, led by Professor Paulo Horta, is looking after the growing process and characterizing the biological activities of the substances extracted. At the Federal University of Paraíba Professor George Miranda is coordinating growing the Gracilaria caudata seaweed species, which is different from those cultivated in Rio Grande do Norte. At the Federal University of Pelotas Professor Marcia Mesko is responsible for biomonitoring and the bioremediation of heavy metals by macroalgae.

Antiviral capacity
Research related to the antifungal, antibacterial, antiviral, anticoagulant and antioxidant activities of algae have been developed by several research groups. In Brazil, professors Valeria Teixeira and Izabel Paixão from the Fluminense Federal University, are conducting studies at the preclinical phase of compounds isolated from macroalgae with antiviral capacity, while Professor Paulo Mourão and Yocie Valentin’s group, from the Federal University of Rio de Janeiro, have isolated polysaccharides with an anticoagulant capability, as presented in a workshop on marine biodiversity, sponsored by FAPESP in September this year.

Besides research into new drugs and bioactive substances of commercial interest, Colepicolo’s group is studying how to use the biomass of algae to produce biodiesel and ethanol. There are two areas of research for obtaining biofuels, one from lipids (fats) and the other from polysaccharides (sugar), extracted from algae. The research involves both macroalgae and microalgae, which cannot be seen with the naked eye. Angela Tonon, a post-doctoral researcher at USP, is molecularly transforming some sugar synthesis pathways into the lipid synthesis pathways from macroalgae. It is a way of having constant extraction, since microalgae are harvested every two or three days, while macroalgae take about three months. Researcher Richard Sayre, director of the Erac Institute for Renewable Fuels, a research center supported by private initiative in Saint Louis, in the United States, keeps in close touch with the research being carried out at USP with regard to the molecular modification of microalgae for producing lipids.

The other way of obtaining biofuel is by the degradation of algal polysaccharides into monosaccharides. The advantage of algae in relation to sugar cane biomass is that there is no need to break down the lignin and other fibers to bring about enzymatic degradation. Researchers are currently selecting efficient yeasts and enzymes that degrade the polysaccharides of macroalgae to produce ethanol. The search for and investigation of new organisms include the isolation of fungi from macroalgae from different locations. “The Antarctic region, where algae up to five meters long live under extreme conditions, will also be considered,” says Colepicolo, who is the coordinator of a project recently approved by the Ministry of Science and Technology and by the Brazilian Navy, within the Brazilian Antarctic Program. “A group of 12 researchers on the project is going to Antarctica in December to collect seaweed.” The proposal is not to carry out bioprospecting, because there is no possibility of large-scale cultivation of this seaweed outside the Antarctic environment. “In addition to characterizing the strains that exist there, we’re going to study the yeasts and fungi that live in symbiosis with these algae,” he reports. It is expected that these microorganisms can be used in bioethanol fermentation processes.

The projects
Bioprospecting studies of marine macroalgae, the use of algal biomass as a source of new drugs and economically viable bioactive substances and their application in repairing impacted areas (marine biodiversity) (nº 2010/50193-1); Type Thematic Project – Biota; Coordinator Pio Colepicolo Neto – USP; Investment R$776,576.35 and US$320,746.40 (FAPESP)
2. Marine algae from the Brazilian coast: isolation and characterization of bioactive substances with potential use in cosmetic formulations. (nº 2003/08735-8); Type Pite – Technological Innovation Partnership Program; Coordinator Pio Colepicolo Neto – USP; Investment R$190,614.03 (FAPESP) and R$170,000.00 (Natura)

Scientific article
CARDOZO, K. H. M. et al. Metabolites from algae with economical impact. Comparative Biochemistry and Physiology. v. 146, p. 60-78. 2007.