The increase in energy demand and advances in biofuel production have drawn increased interest into studies on the use of microalgae as potential sources of third generation biofuels [20].

Considering the state of the art of microalgae technology and today oil prices, algae cannot currently be a considered representative alternative for fossil fuels in the near future [21]. One of the most relevant bottlenecks is the high cost of infrastructure investment and downstream operations such as concentration, dehydration, transformation and purification. The cost of production for the oil and biodiesel from microalgae is estimated to be USD 3.46 / L and USD 3.69 / L respectively. [22]. By contrast, biodiesel obtained from other crops is less expensive such as soybean biodiesel (USD 0.57 / L - USD 0.67 / L), Fischer-Tropsch biofuels from corn fiber (USD 1.13 / L - USD 1.28 / L) and rapid pyrolysis biodiesel from wood chips (USD 0.58 / L) [22,23].

As a result, several efforts have been made to develop faster and cheaper transformation processes. These technologies are mainly focused on optimization of energy and nutrient utilization efficiency, as well as modification of the cellular characteristics and growth conditions to simplify downstream operations. Although some traditional technologies are well known today, we will briefly describe some of the new approaches that are currently under development.

Hydrocarbons are very stable compounds and a substantial source of energy in the world, most of these are naturally found as petroleum [46,47]. However, some studies have shown that cyanobacteria, red algae and dinoflagellates produce noticeable level of hydrocarbons, finding quantities close to 6% of their dry weight. [48-52]. Of particular note, green algae have been shown to have potential for the production of hydrocarbons [50], especially the microalga Botryococcus braunii, which can contain up to 86% of hydrocarbons in dry weight basis [53]. B braunii has the capacity to produce bio-hydrocarbons similar to petroleum, capable of being transformed into high octane gasoline through hydrocracking processes [54,55]. This strain is classified into three different races according to the type of hydrocarbon it produces. Race A produces odd carbon chains (C₂₃-C₃₃) n-alkadienes (mainly diene and triene hydrocarbons); race B produces triterpenoid hydrocarbons, such as botryococenes (C₃₀-C₃₇) and methylated squalene (C₃₁-C₃₄), while race L produces licopadiene which is a C₄₀ tetraterpene [56].

Diesel, gasoline and jet fuel are mainly composed of C₄-C₂₃ alkanes due to their high energy content and hydrophobicity. One of the approaches to producing useful hydrocarbons from microalgae is based on the isoprene molecule. This short-chain volatile hydrocarbon is derived from the first products of the Calvin cycle of photosynthesis, and is produced at substantial rates under certain conditions of environmental stress [57]. The heterologous expression of isoprene synthase in combination with the enzymes of the mevalonic acid pathway has improved the isoprene yield of approximately 2.5 times, compared to that measured in the cyanobacteria transformed only with the isoprene synthase gene. [58].

Another way analyzed by some authors to increase hydrocarbon production is to induce microalgae to produce ethylene. Sakai et al. (1997) studied the conversion of CO₂ to ethylene using Synechococcus PCC7942 expressing the ethylene-forming enzyme (EFE) of Pseudomonas Syringae phaseolicola PK2. This work showed that this recombinant cyanobacterium was capable of producing ethylene and 5% of the total carbon incorporated by the cell was transformed into this hydrocarbon.

Regarding the practical evaluations of the fuel obtained from microalgae, there are limited examples that can be mentioned. The company Solazyme has successfully tested the fuel derived from microalgae in real flights. Continental Airlines flew a plane using a fuel mixture with 40% bio-jet microalgae. In addition, the United States Navy tested a mixture with 50% microalgal fuel in a helicopter [59].

The use of microalgae for agricultural applications is focused on the in vivo use of cyanobacteria, which have a high ability to fix atmospheric nitrogen (N₂) and dispose it in the form of NH₃ for the direct assimilation of plants. Although few microalgae have the capacity to fix this nitrogen, their biomass can be used as an ecological input for agricultural crops. In addition, microalgae release growth promoting substances such as auxins and cytokinins, while also solubilizing phosphates, and the inactive biomass provides nutritious organic matter that improves soil fertility and crop quality. The biomass of microalgae can be applied directly, although the previous hydrolysis allows to improve the positive effects of these products on the plants [96]. In flooded crops, especially rice, the positive effect on production attributed to the presence of the freshwater fern Azolla and the cyanobacteria Anabaena azollae that develop a symbiotic relationship is well known. The annual fixation capacity of atmospheric nitrogen of this system is 350 - 670 kg N fixed / ha, about 12 to 20 times higher than the fixing capacity presented by A. azollae isolated [89,90].

Wuang et al. (2016) used the biomass of Spirulina platensis as an agricultural biofertilizer in different types of crops. In this work it was observed that the application of Spirulina biomass caused an increase in the growth (in dry weight) of the plants A. rugula and red spinach in 21.1% and 155.8% respectively, but for Chinese cabbage did not show differences compared to the control [99]. Overall the quality for use as fertilizer depends on the properties of the microalgal biomass and the nutritional requirements of the plant. In order to meet this specific requirement for each plant, the mixture of biomass from different microalga strains has been proposed. This strategy has been useful and effective in achieving the increase in plant growth. In other studies, González et al. (2016) took advantage of the residual water of the microalgae culture, and the cellular extracts obtained after cell disruption and the microalgal biomass was shown to increase germination rates, as well as growth and production of flowers of the tomato plant (Solanum lycopersicum) [100].

Algae and human nutrition have been related for centuries, the first record found in South America dates from 13,000 years ago in Chile, where algae were used as food, and their medicinal use was known [101]. Table 1 illustrates the biochemical characterization of some foods and biomass of different microalgae species. The protein content, carbohydrates and lipids in microalgae depend on each species and the culture conditions. Its comparison with the nutritional value of daily food is favorable in terms of quality, quantity and protein intake. Likewise, the profile of fatty acids present in the microalgal biomass is similar to that found in higher plants, which are essential fatty acids for human consumption [102]

Table 1

Due to its high protein content, which can exceed 70% in dry weight, vitamin B12, minerals and a high content of ɤ linoleic acid, Arthrospira platensis (Spirulina) is the most commercialized microalgae species cultivated for use as a dietary supplement [103]. The annual production of Spirulina is greater than 5,000 t and the main producers are Japan, USA, India and China [95,96]. In the group of Chloropyceae, Chlorella vulgaris is the most exploited strain due to its high content of protein (50%), vitamins, carotenoids and β-glucan (immunostimulant). The biomass consumption of these microalgae has gained great interest worldwide as food to treat malnutrition. For its therapeutic properties, Spirulina is used for the treatment of cardiovascular diseases, arthritis, anemia and cancer, whereas Chlorella is used to treat problems such as gastric ulcers and neurosis [106].

Currently, there is an emerging market of functional foods from microalgae where a biomass or extracted active substance is added to a food matrix to confer additional nutritional characteristics [107].

Batista et al. carried out various studies on the incorporation of microalgal biomass in different foods such as cookies, pasta and mayonnaise. For example, in one of their investigations, it was observed that the addition Chlorella vulgaris and Haematococcus pluvialis biomass with high carotenoid content increases the oxidative resistance of the mayonnaise lipids and has an emulsifying effect [99,100].

The use of the different microalgal strains as nutritional products is limited by the legal regulations that must be met so that they can be commercialized. Currently, A. platensis and Chlorella sp. are currently the only strains globally allowed to be used as food. However, H. pluvialis and Dunalliella sp. pigments are also produced for use as functional foods. It is expected that in the near future a wide range of strains of microalgae with different nutritional characteristics will be available to expand their use in this field [111].

In animal feed market, especially in aquaculture, the most commonly used strains are Chlorella, Tetraselmis, Isochrysis, Pavlova, Phaeodactylum, Chaetoceros, Nannochloropsis, Skeletonema y Thalassiosira [103,104].

The main role of pigments in microalgae is the light absorption, which is useful for photosynthesis, in addition they can also provide photoprotection against high light intensity. Among the carotenoids produced by microalgae we can find astaxanthin, β-carotene and xanthophylls such as lutein and fucoxanthin. The most common application of natural pigments relies on food industry due to the benefits for human health related to their antioxidant and pro vitamin A properties, and in lesser amounts are used in the pharmaceutical, textile and cosmetic industries [104]. The global market of carotenoid is covered about 50% by astaxanthin and β-carotene.

Haematococcus pluvialis and Dunaliella salina are a worldwide primary source of pigments [107]. H. pluvialis is cultivated in two stages, first under ideal conditions for growth and the second under stressful environmental conditions to induce secondary pigments accumulation [114]. It has been reported that this microalga can produce up to 50 mg / g of astaxanthin under light stress, yielding between 0.5% up to 5% of its dry weight [98,106]. Some authors proposed a single stage process with controlled conditions of nutrient supply, especially nitrogen. However, this process has not yet been carried out on a commercial scale. [107,108]. The price of natural astaxanthin for human consumption exceeds the USD 6.000/kg [17,109].

Similarly, Dunaliella species accumulate high amounts of β carotene, about 10-14% of their dry weight, and have been shown to increase production in response to high salinity and light intensity [105]. Most of Dunaliella cultures are grown in open tanks, taking advantage of their high tolerance to high salt concentrations, which reduces the risks of contamination, and Israel, Australia and the EE. UU are the leading countries in large-scale biotechnological production of β-carotene from microalgae [119].

In relation to lutein, its global market in 2010 was USD 233 million and is likely to increase to USD 308 million by 2018 [120]. Green microalgae produce considerable amounts of this pigment. Chiu et al. (2016) determined that 180 kg of lutein can be obtained annually from 33 tons of biomass / ha of the thermotolerant strains Desmodesmus sp. and Coelastrella sp. grown in open systems [121]. Scenedesmus almeriensis showed a fast growth and high content of lutein for food use and can be produced in closed systems [122], although production in open systems has also been demonstrated [123].

Phycobiloproteins are proteins linked to phycobilins, an accessory photosynthetic pigment, forming a fluorescent active compound present in cyanobacteria and red algae with high potential for application in pharmaceuticals and cosmetics due to its anti-carcinogenic, anti-inflammatory and neuronal protective properties. Phycocyanin (blue) is the main pigment found in Spirulina and phycoerythrin is generally found in rhodophyta Porphyridium. The accumulation of phycobiliproteins in microalgae occurs due to cellular stress generated mainly by light and carbon source availability, in Spirulina platensis the content of phycocyanin varies between 0.11 to 12.7% of the dry weight [124]. These proteins are industrially used as fluorescent marker and in other medical applications [124, 125].

Polyunsaturated fatty acids (PUFAs), especially long-chain fatty acids such as eicosapentaenoic acid (EPA C20:5) and docosahexaenoic acid (DHA C22: 6) are considered essential for humans for being key molecules in health. Due to the low conversion efficiency of the long chains of the omega-3 family, which is about 5% for EPA and less than 0.5% for DHA, whose efficiency decreases even more with age, these fatty acids must be supplemented in the diet [115,116]. Marine microalgae are the primary producers of omega-3 polyunsaturated acids, which are transferred to other organisms through marine food chains. Compared to fish oil, microalgae oil has similar amounts of omega-3, with the advantage of having three times less cholesterol content, has no problems of contamination with heavy metals and toxins, its cultivation is simple and there are some strains that can produce and accumulate a single type of PUFA in a large percentage, facilitating its extraction and purification [117,118]. For example, Crypthecodinium cohnii grows rapidly under heterotrophic conditions, especially when glucose is used as a carbon source and accumulating between 30-50% of DHA dry weight [130].

So far the use Crypthecodinium cohnii and Schizochytrium sp. are the primary sources of commercial DHA which is sold as a supplement in infant formulas [95,120]. Phaeodactylum tricornutum ans Nannochloropsis sp. accumulate EPA, but their commercial production is still not very consolidated. The global market for PUFAs, especially DHA and EPA, is growing exponentially [132]. The consumption of DHA / EPA functional products, especially in regions such as North America, Europe and Asia, represented USD 25.4 billion in 2011 and USD 34.7 billion in 2016 [133]. The price per kg of DHA varies between USD 2,000-3,000, offsetting the high processing costs for the production of microalgal DHA [17].

The polysaccharides obtained from microalgae have a strong potential for use in biodegradable polymeric materials or natural bioactive products that have shown benefits for human health such as anticoagulants, antithrombotic, antitumor and immunomodulatory agents [134]. Microalgae can produce a large amount of polysaccharides during their life cycle and secrete them into extracellular medium, and in combination with other biomolecules such as proteins, lipids and nucleic acids, providing the backbone for protective biofilms. These exopolysaccharides (EPS) can be recovered from supernatants after concentration of biomass, without generating any additional waste, constituting another byproduct that can be obtained under the concept of biorefinery [123,124]. The structure, physical and chemical characteristic of said polysaccharides determine their biological activity and final application. In the majority of microalgae species, depending on the culture conditions, the synthesized polysaccharides are heteropolymers composed mainly of a combination of sugars such as glucose, xylose and galactose, among others. [132]. For this reason, there is a broad spectrum of bioactive compounds with different molecular weights, ionic charges and properties that can have a high value application in various industries such as mechanical engineering, food or biomedicine.

Additionally, Chlorella vulgaris and Gyrodinium impudicum have been reported for their ability to generate homopolysaccharides composed of glucose as part of their cell wall and galactose exopolysaccharides, respectively.

Under autotrophic conditions, microalgae use light and inorganic carbon as sources of energy and carbon, respectively. However, some species can grow mixotropically, assimilating organic carbon sources as well, resulting in higher biomass [129,130]. In spite of this last advantage, traditional organic sources (sugars, organic acids, glycerol etc.) turn out to be costly alternatives if they are used in their pure form and could compromise the quality of the open systems due to the proliferation of contaminants [141]. In that sense, the use of inorganic carbon salts, the use of combustion gases and the implementation of systems coupled with nutrient-rich wastewater treatments have been proposed [131,132].

Some authors have evaluated wastewater from pig farms, as low-cost raw materials, as well as effluents from breweries, and by-products and wastes from the food and agroindustrial industry such as cane molasses, silage juice, serum permeate, among others [129,130,133]. These waste streams have been satisfactorily evaluated and are emerging as low-cost raw materials in the preparation of culture media for microalgae [134,135].

Nitrogen is the second most important nutrient for microalgae biomass proliferation, its cellular content can vary from 1% to 14% on a dry weight basis [147]. Algae can assimilate inorganic nitrogen molecules such as NO₃ ^-, NO₂ ^-, NO, NH₄ ⁺ and some cyanobacteria, like Oscillatoria sp., Nostoc sp., Anabaena sp., and some diatoms like Rhizosolenia sp. and Hemiaulusare sp. can fix N₂. They can also assimilate N in organic forms, such as urea, peptone, yeast extract, amino acids and purines [136,137]. In urban wastewater, urine provides 70% nitrogen, 40% phosphorus and 60% potassium of the total load and represents approximately 1% of the volume of water [138,139]. This nitrogen/phosphorous rich residue has been used for cultivation of Arthrospira platensis and Chlorella sorokiniana.

Markou et al. (2014) proposed a novel alternative ammonium nitrogen recovery using zeolites as an adsorbent material in wastewater with a high content of nitrogen, dissolved solids, dyes and recalcitrant compounds that hinder the passage of light, which inhibit the growth of microalgae [142]. In a subsequent study, Markou et al. (2015) determined that the microalgae A. platensis and C. vulgaris can grow with the nutrients provided by the ashes of hatchery residues of birds with high content of phosphorus, potassium, and ammonium recovered from the process of drying this residue. Simultaneously, they proposed the use of these microalgae biomass as feed for birds, creating a closed production system [151].

Phosphorus is an essential element for different cellular processes like energy storage and transfer in the form of ATP, as well as biosynthesis of nucleic acids, phospholipids, among many other [148]. The content of this element in the algal biomass is generally from 1.0 to 3.3% by dry weight. The traditional sources of phosphorus at the laboratory level are mainly inorganic salts such as K₂HPO₄, KH₂PO₄, K₃PO₄, (NH₄)₃PO₄. Although phosphorus is a minor component of the biomass of microalgae, it is still necessary to identify and evaluate new alternatives for recovery and use of this element, taking into account that for this particular nutrient, there is no a biogeochemical mechanism that recirculate it at the level of the biosphere, in contrast of what happens with carbon and nitrogen [57].

Mukherjee et al. (2015) developed a method of recovering and recycling phosphorus from wastewater generated during rice parboiling using microalgae. They isolated Cyanobacterium sp., Lyngbya sp., Anabaena sp. and Chlorella sp. in eutrophic environments and used them as bioremediation agents. After harvesting the biomass, they used them as a biofertilizer to plant rice seedlings and observed that the release of phosphorus by the action of solubilizing organisms is similar between biofertilizer and chemical fertilizer. [152].

Moed et al. (2015) investigated the use of struvite (MgNH₄PO₄) as a source of nitrogen, phosphorus and magnesium for the cultivation of C. vulgaris. In the tests they used 721 mg / L of struvite for the Bold Basal Medium (BBM). This modified medium can produce 2.85 g / L of lipids suitable for the production of biodiesel, comparable to the use of BBM without struvite [153].

These are elements that appear in very small quantities, on the on the micro-level (μg/L), or parts per billion (ppb). These trace elements act as enzymatic cofactors and are important for the biosynthesis of many cellular compounds. The essential micronutrients for microalgae are mainly Mg, S, Ca, Na, Cl, Fe, Zn, Cu, Mo, Mn, B and Co. Many wastewater and industrial by-products all or a large number of these micronutrients and for this reason they are explored as raw material for the cultivation of microalgae. [131,137].

Considering the processes of obtaining biofuels derived from microalgae, part of the sustainability of the process lies on the favorable nature of the energy balance [154]. Throughout the process of biomass production and byproducts, a series of operations are involved related to incubation, harvesting, processing and purification [155]. The highest proportion of energy consumption is associated with aeration, pumping, centrifugation, harvesting and drying [156].

Shimako et al. (2016) investigated two bioenergy production systems involving biodiesel and biogas, where electricity and heat are generated. These authors found that the net energy balance was negative for the biodiesel production process. However, the energy balance in the biogas production process was positive, which suggests that this could represent an alternative to establish the process to a greater scale [157]. Doušková et al. (2010), proposed a novel process for the use of organic liquid waste in order to simultaneously produce energy and microalgae. They used vinasse as a substrate for the production of biogas in an anaerobic fermentor and later the biogas was fed to photobioreactors as a source of CO₂ for the cultivation of microalgae. Once the biogas is produced, the wastewater from the anaerobic fermentor is processed to obtain an ammonium-based fertilizer that can be used directly as a nitrogen source in the culture medium of the microalgae or for the production of ammonia liquor [158]. Shimako et al. (2016) investigated two bioenergy production systems involving biodiesel and biogas, where electricity and heat are generated. These authors found that the net energy balance was negative for the biodiesel production process, however, the energy balance in the biogas production process was positive, which suggests that this could be the alternative to establish the process to a greater scale [157]. Doušková et al. (2010), proposed a novel process for the use of organic liquid waste in order to simultaneously produce energy and microalgae. They used vinasse as a substrate for the production of biogas in an anaerobic fermentor and later the biogas was fed to photobioreactors as a source of CO₂ for the cultivation of microalgae. Once the biogas is produced, the wastewater from the anaerobic fermentor is processed to obtain an ammonia-based fertilizer that can be used directly as a nitrogen source in the culture medium of the microalgae or for the production of ammonia liquor [158].

Sevigné Itoiz et al. (2012), analyzed the life cycle and energy balance in the production of marine microalgae biomass, finding that in closed systems, the total energy requirement was 923 MJ kg^-1, whereas for outdoor cultures it was 139 MJ kg^-1. Additionally, these authors affirm that during cultivation, the highest energy consumption occurs in the growth stage. Although the energy balance was negative for both systems under the conditions studied, the authors suggest that outdoor cultivation could be optimized to obtain a positive balance considering the use of raceway or flat panel reactors [159].

In addition to the energy required for the operations described above, light energy can also be considered as a critical input concerning the energy balance. The primary source of PAR (Photosynthetic Active Radiation) photons is sunlight, which is free and available throughout the year in some regions. However, sudden variations in irradiation and change in weather and seasonal conditions can be detrimental for production. In contrast, the use of artificial sources such as fluorescent tubes, high intensity discharge lamps (HID) and LED lights, offer continuity and stability in the energy supply but can represent prohibitive costs in terms of investment and operation in massive cultures [154]. Under conditions of artificial lighting, Blanken et al. (2013) found that the energy demand during the process can be compared with the enthalpy of combustion of algae biomass (0.477 MJ C-mol^-1). They concluded that a PAR light efficiency of 2 μE s^-1 W^-1 and a biomass/light yield of 1,0 a 1,5 g-DW E^-1, only 4% to 6% of the electric energy supplied initially is conserved in the biomass, so that a large amount of energy is lost in cultures with artificial light and the use of this should be avoided. However, the electrical energy required for the cultivation with artificial light could be generated as 'green' energy by photovoltaic, wind or geothermal [154].

The light quality influences cell growth and biochemistry, so artificial lighting allows manipulation of the final biomass for specific uses. The use of artificial light ensures a better regulation of the flow density of photosynthetic photons, of the photoperiod and the light spectrum in the production of microalgae. LED light can also be used to adjust the biochemical composition of microalgae biomass through individual wavelengths at different intensities or frequencies of light pulses [160]. However, the implementation of artificial lighting feasibility to produce microalgae will be subject to the improvement of the energy efficiency of the sources and their cost of production, as well as the biological improvement of the photosynthetic efficiency.