Microalgae has a great potential to produce biofuels and bioproduct but the cost is still too high mainly due to the biomass production. Mixotrophic cultivation has been pointed as microalgae cultivation mode for biomass/bioenergy production with lower cost and able to make remediation of organic waste. The proposals of this work was to make a review of microalgae growth under mixotrophic condition using agro-industrial waste. Agro-industrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. However biotechnological technologies are necessary to develop the production of microalgae on a large scale.
- mixotrophic growth
- agro-industrial waste
Microalgae are a variety of autotrophic, prokaryotic or eukaryotic organisms, where their single-cell structure allows solar energy to be easily converted into chemical energy. This biochemical conversion is being used commercially to obtain the biomass, consequently, in the insertion in products with commercial application. The most used microalgae cultivation techniques are opens aerated lagoons and closed photobioreactors [1, 2, 3, 4].
Due to the advantages that microalgae offer over many other species, researchers and entrepreneurs have shown great interest in the development of production processes for biofuels, functional foods and bio-products from different species. Compared to terrestrial crops, these microorganisms have photosynthetic efficiency, growth rate and higher biomass production, consequently mass cultivation for commercial microalgae production can be carried out efficiently . In addition, the cultivation of microalgae does not require arable soil, and can be grown in saline, brackish and wastewater and in harsh conditions, not competing with the production of food that is currently a major challenge for the production of first and second biofuels generation . Therefore, competition for arable land with other crops, especially for human consumption, is greatly reduced.
Although most microalgae grow exclusively through photosynthesis, some species are mixotrophic and use extracellular organic carbon when a light source is not available . Microalgae can be a source of several important compounds, including hydrogen and hydrocarbons, pigments and dyes, food and feed, biopolymers, biofertilizers, insecticides, neutraceuticals (foods capable of providing health benefits) and pharmacological compounds, in addition to being a potential biomass for production of biofuels .
Although the production of microalgae does not directly compete with food production and can be grown in harsh conditions, economic viability does not yet exist in many of the processes of industrial interest. However, the improvement and mastery of technologies capable of making inserted industrial processes viable become essential. Despite of the microalgae have a wide potential for production and applications, there are many obstacles to the biodiversity of these algae, such as mastery of technologies for production, genetic improvement research of strains more resistant to pathogens and economic viability in large-scale production [9, 10]. According to Georgianna and Mayfield , although promising, the success of inserting microalgae in the production of various products depends mainly on two important factors: high productivity and quality of biomass, as well as cost-effective production.
One of the viable solutions to reduce the costs of microalgae biomass production is to explore different forms of energy metabolism, highlighting the photoautotrophic, heterotrophic and mixotrophic for commercial production. Understanding these forms of metabolism allows the application of efficient crop strategies aimed at increasing the production of biomass and bioproducts on a large scale with cost optimization to couple the agroindustry waste treatment . Microalgae are able to eliminate a variety of pollutants in wastewater mainly nitrogenated, phosphates and organic carbons .
Mixotrophic cultivation is a preferable microalgae growth mode for biomass production . Compared to photoautotrophic and heterotrophic metabolism, mixotrophic cultures have been demonstrated many advantages, such as less risk of contamination, reduced cost and high biomass productivity. Even susceptible to contaminations, the use of photobioreactors minimizes this risk, but increases the cost of the process, which can be offset by the high biomass yield that can reach 5–15 g/L, being 3–30 times higher than those produced under autotrophic growth conditions [14, 15].
The use of waste for microalgae mixotrophic growth has been researched, mainly with the objective of expanding and diversifying in an alternative way the control and combating the inappropriate disposal of these in the respective industries, combined with the perspective of minimizing the operational costs of producing microalgae in large scale that are still considered high. The waste generated by the agribusiness has a high load of organic matter with high concentrations of Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), ammonia, phosphates, suspended solids harmful to the environment, in addition to dissolved components such as sugars, fat and proteins originating from food, contributing to environmental pollution .
According to Patel et al. , research involving the mixotrophic cultivation of microalgae using organic matter as a source of carbon points to the production of high yields of biomass and biocomposites of industrial interest when compared to systems involving photoautotrophic and heterotrophic metabolisms. In this sense, recent studies have been carried out using agroindustrial waste to grow microalgae in a mixotrophic regime in order to minimize the cost of the biomass production process and treat the effluent adding value to the process, suggesting a microalgae biorefinery system .
Patel et al.  cultivated
Xio-Bo Tan et al.  demonstrated that
Wang et al.  utilized glucose recovered from enzymatic hydrolysis of food waste as culture medium in mixotrophic cultivation of
Due to the success of mixotrophic microalgae growth, the use of agro-industrial by-products stands out, adding value to production processes and reducing costs. The nutritional characteristics, availability and low cost of obtaining evidence the possibility of using the by-products in the cultivation of microalgae. This work reviews the mixotrophic cultivation system of microalgae using waste from agribusiness as a source of organic carbon, pointing out the benefits of this strategy as a solution to the environmental problems caused by these effluents, adding value to an industrial process for the production of biomass and biocompounds as biorefinery.
Microalgae is a generic term used to refer a widely diverse group of photosynthetic microorganisms . There are several species of microalgae, which are found in aquatic environments of fresh water, brackish and saline . Microalgae in general, have varying microscopic sizes, perform photosynthesis, use carbon dioxide as a nutrient source for growth, in addition to playing a fundamental role in ecosystems [23, 24, 25]. It is estimated that there are about 800 thousand species of microalgae, of which about 40 to 50 thousand are of scientific knowledge, which makes it an almost unexplored resource, demonstrating the great biodiversity of these algae [26, 27]. In addition, most species are not yet known and very few are used for any purpose.
The basic composition of microalgae is based on carbohydrates, lipids, proteins, ash and nucleic acids, in addition to chlorophyll and other protective pigments and light capture that provide high photosynthetic capacity, allowing conversion of up to 10% of energy in biomass . In conventional plants, this percentage is higher when compared to other conventional plants, whose conversion is limited to a maximum of 5% . The predominant elements in the biomass of microalgae are carbon, nitrogen and phosphorus and some metals such as iron, cobalt, zinc is also found .
3. Application of microalgae
In recent years, several researches have been carried out seeking to develop technologies for the elaboration and diversification of products based on microalgae. The growing expansion of these products is part of a wide range of utilities inserted in the most different commercial niches, expanding the possibilities of use and adding value to the market. According to Hu et al. , the global algae market is expected to be worth of about $ 1.1 billion by 2024.
Microalgae are inserted in a wide variety of species, distinguishing one from the other due to their biological structure. In this sense, these microorganisms offer potential possibilities for CO2 biofixation, remediation, effluent treatment, production of biofuels, high-value products including pharmaceuticals, food and neutraceutics .
According to Rizwan et al. , microalgae can be a source of antioxidant compounds, carotenoids, enzyme polymer, lipid, natural dye, polyunsaturated fatty acid, peptide, toxin and sterols, which are widely used in industry. In addition, they are used for the synthesis of antiviral, antimicrobial, antiviral, antibacterial and anticancer drugs .
Commercial microalgae cultivation systems are operated to produce mainly pigments and metabolites for nutritional supplements . The algae that have technical and economic viability of production are Spirulina (Arthrospira) for supplements with a high protein content, Haematococcus as a source of astaxanthin and
Spirulina consists mainly of proteins (50–70%), being widely used in human nutrition to combat malnutrition . This species is rich in essential amino acids, beta-carotene, minerals, essential fatty acids, vitamins, polysaccharides, among others. Chlorella accumulate high concentrations of carotenoids (astaxanthin, lutein, β-carotene, violaxanthin and zeaxanthin), antioxidants, vitamins, polysaccharides, proteins, peptides and fatty acids [5, 38]. In addition to all the benefits mentioned, the bioactive compounds of microalgae can have a biological, immune, antiviral and anti-cancer properties, being highly active .
Global warming has been worrying environmentalists across the planet. Although there are different ways of capturing CO2, the biological method stands out as a potentially attractive alternative. The requirements for producing and obtaining biomass from microalgae are basically CO2 and a source of light, be it natural or artificial . Carbon dioxide can be converted into organic matter by performing photosynthesis using sunlight as an energy source [40, 41, 42]. Microalgae are more efficient for fixing CO2 and have a higher productivity rate (ton/ha/year) when compared to terrestrial plants. In addition, CO2 biofixation can be combined with other processes, such as the treatment of organic waste, being advantageous in terms of economic viability and environmental sustainability. Microalgae can also be grown in nutrient-rich organic effluents, salt and brackish water, reducing the use of fertile land and fresh drinking water .
Studies involving the mixotrophic cultivation of microalgae using industrial residues from agro-industry as a source of organic carbon have been carried out to minimize the cost of biomass production, treat the effluent and promote CO2 biofixation . In this sense, expanding the ways in which these residues are used, avoiding their incorrect disposal, minimizes the effects of environmental pollution and adds value to industrial processes, encouraging a cleaner and more sustainable bioeconomy.
Currently, fossil fuels represent the main source of energy in the world, but unsustainable and directly related to the pollution of air, land, water and climate change. The burning of fossil fuels consolidated to increase the atmospheric concentration of CO2 being directly associated with global warming. Allied to this, the future oil scarcity is a major challenge for scientists, motivating a constant search for technologies capable of producing clean and sustainable fuels . Among many biomasses, microalgae represent a promising source for the production of clean renewable energy, as they are capable of fixing CO2 by performing photosynthesis with efficiency and productivity superior to that of conventional oilseeds and terrestrial plants used in the production of biodiesel and bioethanol. Among the available biomass sources, microalgae have been evaluated and investigated as generation third biomass, being researched to produce different types of biofuels, among which are biodiesel, bioethanol, bio-oil, char, hydrogen and synthesis gas . Recent research involving the production of biofuels has been focused on third generation biomass, since the first and second raw materials are based on terrestrial cultures that compete with food production and can lead to food crises . Algae biofuels are not yet obtained on a large scale due to the high cost of the process justifying the development of new technologies that can bring economic viability .
Bioremediation and biofuel production from waste resources by microalgae platform is mainly important to utilize abundantly available solar energy biofixing CO2 and treat effluents through the mixotrophic growth of microalgae . Algal bioremediation is a good strategy to produce biomass for biofuels production while remediating wastes, also improving carbon-footprint through carbon capturing and utilization technology.
A microalgae biorefinery enables to integrate fractionation and conversion processes to transform biomass into bioproducts such as food, feed, chemicals, and bio-energy as optimization of the use of the microalgae for reducing waste production, and maximizes process profit. After lipid transesterification for biodiesel, the residual biomass can be used to produce other biofuels such as methane, bio-oil and ethanol or biocompounds for food and pharmaceutical industry .
4. Cultivation systems of microalgae
There are currently four cultivation technologies in use for the production of commercial microalgae including open ponds and raceway ponds (open systems), photobioreactors and fermenters . In open systems, microalgae are grown in open areas, including tanks, lakes, and ponds, deep channels, among others. In closed systems, crops are grown in transparent bioreactors, exposed to sunlight or artificial radiation for photosynthesis and fermenters.
4.1 Opens systems
Natural and artificial lakes and ponds, where most of the systems commonly used are large, shallow ponds and tanks, represent open pond systems. The main advantages of these systems are the ease of construction and operation when compared to photobioreactors and the possibility of operating hybrid processes involving the cultivation of algae associated with the treatment of wastewater. However, the disadvantages are inefficient light distribution, losses through evaporation, diffusion of CO2 into the atmosphere, contamination and the requirement for large areas of land . Open ponds are currently in use for wastewater treatment and production of
Closed or artificial ponds (circular ponds and raceway ponds) are more efficient than open systems for producing microalgae, since control over the production environment is much better than open ponds or extensive ponds. The cost of raceway ponds is higher than that of open lagoon systems, but lower than that of photobioreactors. These systems are the most used due to their potential to produce large quantities of biomass for commercial application. Raceway pond ponds are commonly used to grow
4.2 Closed systems
Photobioreactors were designed to overcome the problems associated with open growth systems. It has been shown that cultivation of microalgae in these systems are capable of producing large amounts of biomass as they allow an effective control of process parameters, such as pH, temperature, CO2 concentration, level of contamination, among others. However, photobioreactors are much more expensive than open ponds and raceway ponds. Commercial photobioreactor productions include the production of
Closed fermenters are used for the production of heterotrophic algae, where sugars or other simple carbon sources are used for growth instead of CO2 and light. Open fermenters similar to the fermenters used in the production of ethanol in industries are not suitable for the growth of algae, since these microorganisms have slow growth when compared to yeasts and bacteria. In the USA, India and China ω-3 fatty acids are produced from
5. Growth metabolism
Microalgae have different growth metabolisms, which characterizes their versatility. Cultivation conditions define the metabolic route for the production of biocompounds, including proteins, carbohydrates, pigments and fatty acids. Although the production of microalgae has traditionally been photoautotrophic, these microorganisms have different forms of energy metabolism including heterotrophic and mixotrophic, which use an organic carbon source for the growth and production of biomass. The understanding of these metabolisms allows the diversification of current growth systems aiming at increasing the production of biomass and certain specific metabolites.
Photoautotrophic growth is the most common way to cultivate microalgae through photosynthesis. In these systems, high concentrations of CO2 are sequestered, but productivity is low when compared to heterotrophic and mixotrophic systems that provide high yields of biomass and secondary metabolites [51, 52]. Through photobioreactors is possible to obtain the maximum cell density of 40 g/L, while in outdoor open-pond or raceway-pond cultures, the cell concentration is usually lower than 10 g/L. This significantly increases the energy consumption of cell harvesting and the cost of biomass production . Usually scale-up of microalgae cultivation in wastewater is fulfilled phototrophically, which may be hindered by inefficient illumination and the low biomass density, leading to poor removal of nutrients .
For photosynthesis, light is used as an energy source and CO2 is used as a carbon source . The carbon source is essential for growth and the higher its concentration, the higher the productivity. Nitrogen sources in the culture medium are also essential for the synthesis of proteins, nucleic acids and other biocomposites necessary for cell growth and survival  and the concentration must be compatible with the amount of carbon in the medium . The type of cultivation, the nutrients, the carbon source, the salinity of the medium, the irradiance and the temperature vary according to the chosen species and also considerably influence the success of the microalgae production.
Microalgae can grow in the absence of light in culture media assimilating organic carbon. Studies show that some species, including
In heterotrophic crops, microalgae acquire carbon and energy from organic sources via oxidative phosphorylation, consuming O2 and releasing CO2. According to Behrens , the cost of producing the kg of dry biomass produced in photoautotrophic conditions can be 5.5 times higher than in heterotrophic conditions. In heterotrophic systems there are no light limitation problems, since microalgae grow in the absence of light using organic carbon as a carbon and energy source reaching high concentrations of biomass reaching up to 100 g/L, which considerably facilitates the harvesting process . In heterotrophic processes, 18% of the energy obtained can be converted into adenosine triphosphate (ATP) whereas in photoautotrophic cultures this percentage is only 10% . However, despite the advantages, the cultivation of microalgae under heterotrophic conditions, due to the use of organic carbon, requires reactors and techniques of greater complexity and high cost to avoid contamination caused by other microorganisms.
Under mixotrophic conditions, microalgae grow both photoautotrophically and heterotrophically, being able to assimilate organic compounds as a carbon source and use inorganic carbon as an electron donor . In this metabolism, the energy is captured through the catalysis of external organic compounds through respiration and the light energy is converted into chemical compounds via photosynthesis, being a promising solution in the processes of environmental remediation, being able to treat the effluents of the agribusiness and produce biomass rich in metabolites of industrial interest.
In photoautotrophic crops, self-shade caused by the high density of cells makes light penetration difficult, causing photoinhibition and may be the limiting factor to its propagation leading to low biomass yields. Under heterotrophic conditions, not all microalgae species are able to grow and the strict use of only organic substrates as a source of carbon and energy makes the process more prone to contamination. Thus, an alternative to maximize production can be through mixotrophic cultivation. Therefore, the cells would multiply with autotrophic metabolism until reaching the maximum cell density, at which point a source of organic carbon is added to stimulate, also, heterotrophic growth. Thus, due to the high cell density in the medium, problems with contamination of microorganisms would be less likely to happen. For the success of this technique, the cultivated species must be able to grow in heterotrophic conditions without microbial contamination and the organic compound to be added, as well as its ideal concentration must be known.
Reducing use of light in mixotrophic processes decreases the demand for energy, which minimizes the operational cost in processes with artificial light. According to Perez-Garcia and Bashan  in these systems there is a better control capable of regulating the growth rate of the species, which minimizes the risk of contamination by photosynthetic microorganisms. Due to the significant heterotrophic contribution of the organic fraction, the reactor design does not require a maximized area to expose the microalgae to light to perform photosynthesis, decreasing the process cost.
According to Patel et al. , mixotrophic cultivation has advantages over photoautotrophic and heterotrophic metabolism. In this process, in addition to the higher growth rates reducing the microalgae growth cycle, there is also growth in the dark phase mediated by respiration, potentiating the production of biomass. There is also a comparatively prolonged exponential growth phase, great flexibility to change the metabolism from heterotrophic to photo-autotrophic and vice versa, prevention of photo-oxidative damage caused by O2 accumulated in closed photobioreactors and reduction in substrate uptake photoinhibition. It has been shown that microalgae cultivated under mixotrophic conditions can present, under controlled conditions, higher biomass productivity when compared to photoautotrophic and heterotrophic cultures .
6. Growth microalgae using waste from agro-industry
The advancement of agro-industry has generated a large amount and variety of waste causing serious environmental problems and is one of the sectors that generate more waste rich in organic matter. According to Dahiya et al. , approximately 1.3 billion tons of foods are wasted each year during its production, handling, storage, processing, distribution or consumption. The composition of food processing residues is extremely varied and depends on both the nature of the raw material and the production technique employed.
Due to the scarcity of available areas close to large urban centers for the disposal of industrial and urban waste, the vast majority of companies do not carry out treatment and/or correct disposal of this material, which contributes to the increase in environmental pollution. Pollution is due to high concentrations of organic matter and heavy metals causing contamination, eutrophication of water bodies, death of aquatic organisms and local vegetation, ecological imbalance and health problems for the population. In this sense, the use of agro-industrial waste as a source of organic carbon for the cultivation of microalgae is presented as an option for the bioremediation of these effluents added to the production of biomass rich in biocompounds with different applications.
A lot of studies have evaluated the mixotrophic growth of microalgae using glucose, glycerol and acetate as a source of organic carbon and observed that the biomass yields were higher, which could decrease the cost of the process. Liang et al.  investigated
Due to the success of the microalgae mixotrophic cultivation method, recent studies have been evaluating the possibility of using residues from the agro-industry as source of organic matter as a strategy to treat the effluent, produce biomass and reduce the cost of the process.
Hu et al.  evaluated the mixotrophic cultivation of
Li et al.  investigated the effects of autotrophic and mixotrophic growth on cell growth and lipid productivity of green microalgae
León-Vaz et al.  cultivated
Bhatnagar et al.  evaluated mixotrophic growth of
Andrade & Coosta  determined the effects of molasses concentration and light levels on mixotrophic biomass production by
Melo et al.  evaluated the growth, nutrients and toxicity removal of
Hugo et al.  studied the growth of forty microalgae strains under mixotrophic conditions using sugarcane vinasse as source of organic matter.
Mitra et al.  cultivated
Salati et al.  cultivated
Piasecka et al.  studied the growth of
Tsolcha et al.  evaluated a mixed cyanobacterial-mixotrophic algal population, dominated by the filamentous cyanobacterium
Grupta et al.  cultivated the
Yeesang et al.  evaluated
Gélinas et al.  studied the mixotrophic growth and lipid production of
Nur et al.  studied palm oil mill effluent (POME), one of the wastewaters generated from palm oil mills, as source of organic carbon for mixotrophic microalgae growth. The aim of this research was to identify the growth of
Manzoor et al. 2020 presented the growth of
Microalgal biomass represents a sustainable alternative to fossil consumption and bioproducts for food and pharmaceutical industry. Microalgae can growth under photoautotrophic, heterotrophic or mixotrophic modes where the latter two trophic modes require organic carbon to grow efficiently. Actually, researchers have highlighted the role of low cost-efficient agro-industrial by-products used as supplements in algal culture media. However, supplementation of organic carbon contributes significantly to a higher cost of microalgae production and this can compete with human and animal alimentation. Agro-industrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance.
Faced with this scenario, biotechnological technologies are necessary to develop the production of microalgae on a large scale and expand the range of utilities that, in the short or long term, contribute to the improvement of industrial processes. In addition, mixotrophic microalgae growth is a great strategy to reduce environmental pollution generated by residues rich in organic matter and can reduce the cost of the industrial process. However, more investments, development and greater knowledge of the metabolism of these microalgae and their effectiveness in the generation of new bioproducts are increasingly necessary.
The authors gratefully acknowledge the Postgraduation Dean’s Office of the Federal University of Rio Grande do Norte and Dean of Extension of Federal Institute of Rio Grande do Norte. The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support.