Heterotrophic growth in microalgae is nonphotosynthetic / dark metabolism and involves utilization of organic carbon substrates for energy production and other metabolic activities (Morales-Sánchez et al. 2015). Heterotrophic culturing of microalgae is easier to operate, produces increased cell mass, leads to high growth rates, produces high amounts of metabolites, enables mono-culturing and low cost operation and construction of infrastructure (Perez-Garcia et al. 2011). The heterotrophic metabolic pathway entails three major steps: (1) uptake / assimilation, (2) activation and transformation, and (3) storage / utilization of metabolites (Morales-Sánchez et al. 2015). Uptake of carbon source like glucose requires symporter system–a cytoplasmic membrane bond protein (Morales-Sánchez et al. 2013) but other carbon sources (e.g., glycerol) do not as they easily diffuse into the cell (Perez-Garcia et al. 2011). Then carbon source undergoes glycolysis (Embden-Meyerhof pathway and pentose phosphate pathway), tricarboxylic acid cycle, and glyoxylate cycle pathways. The products are carbon skeleton metabolites such as fatty acid, intracellular polysaccharide, proteins, etc. The details of reactions flow / pathways are presented in Fig. 2. Microalgae used nitrogen for synthesis of amino acids during heterotrophic growth regime. Nitrogen sources for microalgae culturing are ammonium, nitrate, nitrite, urea and amino acids, peptone, purines, and yeast extract, etc. (Chen and Chen 2006). Ammonium is most preferred due to less energy is required for its uptake (Shi et al. 2000). However, several reports have shown that nitrate assimilation in microalgae is inhibited by factors like light (Morris 1974), presence of heavy metals above 150 μM (Devriese et al. 2001) as well as presence of ammonium (Cannons and Pendleton 1994). Urea and other organic nitrogen compounds are also suitable for heterotrophic growth regime of microalgae. Hence, during the growth in wastewater which are rich in organics, probably dark in color and have ample inorganic nutrient present (e.g., agro industry based effluents), microalgae will show heterotrophic regime.

Autotrophic growth of microalgae involves photosynthetic reduction cycle-light reaction, CO₂ concentration, and dark reaction (Kruse et al. 2005). Briefly, microalgal pigments (chlorophylls, phycobilins, and carotenoids) absorb photon to generate electrons, which are transferred in to PSI, II and cytochrome b6f complexes, ADP⁺ and NADP⁺ are converted to ATP and NADPH, respectively. The ATP and NADPH are used to drive biochemical reaction during the dark reaction for CO₂ assimilation through Calvin Benson cycle in the chloroplast (Wang et al. 2014) (Fig. 2). The diffusion rate of CO₂ in water and that from bicarbonate degradation in stroma is very low. Concentration of CO₂ provides adequate amounts of CO₂ to stroma in chloroplast and reduces O₂ hindrance on ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) (Nelson et al. 2008). Microalgae concentrate CO₂ using carbonic anhydrase, inorganic carbon transporters, sequestered RuBisCOs in elevated CO₂ micro-compartment and carboxylation mechanisms (CAM and C₄-photosynthesis) (Giordano et al. 2005, Wang et al. 2011, Baba and Shiraiwa 2012). Microalgal carbonic anhydrases (Table 1) are zinc-metalloenzymes responsible for rapid inter-conversion (CO₂ + H₂O ↔ HCO₃⁻ + H⁺) of inorganic carbon species (Moroney et al. 2011). Dissolved inorganic carbon transporters facilitate uptake of inorganic carbon and CO₂ (Wang et al. 2011). Inorganic carbon transporters (Pollock et al. 2004, Ohnishi et al. 2010) include HLA3 at plasma membrane, LCIA at chloroplast membrane and CCP1 and 2 at chloroplast. Rhesus proteins also function as channels for CO₂ in microalgae (Soupene et al. 2002) and that Rhesus-1 protein is a bidirectional channel for the CO₂ in Chlamydomonas reinhardtii (Soupene et al. 2004). Several wastewaters, such as agricultural runoff, dairy farm effluents, and anaerobically digested manure are rich in inorganic nutrients but have low or no organic carbon (Prajapati et al. 2014a, Choudhary et al. 2015, 2016). For remediation of such wastewaters, addition of external carbon source is crucial, if microbes (other than microalgae), are being used. However, microalgae can efficiently remediate such wastewater without need of external carbon source as they can sequester CO₂ from environment through autotrophic growth.

Range of wastewater contain adequate amount of both inorganic nutrients and biodegradable organic carbon source. In such wastewater, microalgae usually grow in mixotrophically. The mixotrophic growth regime involves simultaneous utilization of both inorganic and organic carbon substrate (Cerón-García et al. 2013). Mixotrophic microalgae have cellular apparatus for heterotrophic and autotrophic metabolism. Mixotrophic culture of microalgae resulted in higher growth rate, yield of metabolites, than auto- and hetero-trophic (Ogbonna et al. 2002, Das et al. 2011). A recent study has reported higher biomass production through mixotrphic microalgal growth wastewater supplemented with various carbon sources (Bhatnagar et al. 2011). Mixotropic is usually a complementary (Ogbonna et al. 2002, Fernández Sevilla et al. 2004) and a synergetic mechanism of auto- and hetero-trophic (Cheirsilp and Torpee 2012). However, other researches have shown that microalgae generate higher energy in heterotrophic growth regime than in the case of mixotrophic condition (Yang et al. 2000, Hong and Lee 2007, Perez-Garcia et al. 2011). There are many internal controls and factors that regulate the rate at which each metabolic pathway proceeds. Microalgae respiration and photosynthesis activities are interconnected as cellular organelles communicate and exchange by-products (Yang et al. 2015), for instance (1) utilization of sugar / polysaccharide produced in chloroplasts through photosynthesis by mitochondria, (2) acquiring of ATP produce in mitochondria by chloroplasts when the rate of ATP generation in photophosphorylation is lower than the rate of ATP utilization in RuBisCO, and (3) utilization and removal of cellular O₂ during respiration in mitochondria thus enhance efficiency of photosynthesis as are result of decrease in possibility of photoreduction by PSI (Dang et al. 2014, Yang et al. 2015). While cultivating microalgae in open ponds or photobioreactor, part of the culture remains unexposed to light. If the adequate amount of organic carbon and nutrients are present, mixotrophic growth may dominant in these regions having no or limited light availability (Ji et al. 2014b, Perez-Garcia and Bashan 2015). However, sometimes it is difficult to differentiate the mixotrphic growth from the synergic growth of microalgae and the native bacteria of the wastewater system (Fig. 1). It is well reported that the microalgae and the bacteria in the wastewater grow in a synergic relationship through exchange of nutrients, CO₂ and O₂ (Rawat et al. 2011, Ramanan et al. 2016). Hence, apart from the mixotrophic growth, synergic growth also results in higher rate of nutrient / pollutant removal and biomass production during bioremediation.

Control of increasing atmospheric concentration of CO₂ is achievable by algal-induced CO₂ fixation (Alpert et al. 1992). Researches in this area focused on strain selection, culture condition optimization, adaptive evolution, and mutation study (López et al. 2013). Microalgae are divided into (1) CO₂ sensitive (intolerance) species inhibited by <2–5% CO₂, (2) CO₂ tolerant species that can cope with 5–20% CO₂, and (3) extreme CO₂ tolerant species that tolerate 20–100% CO₂ (Solovchenko and Khozin-Goldberg 2013). High CO₂ cause high ATP generation, shutdown of CO₂-concentrating mechanisms, upregulation of H⁺-ATPases, alkalization of medium and adjustment of membranes’ fatty acid composition (Solovchenko and Khozin-Goldberg 2013). During autotrophic and mixotrophic processing, increase in CO₂ concentration in medium caused microalgae to accumulate lipid and polyunsaturated fatty acid (Tang et al. 2011) and stop organic substrate utilization (Sforza et al. 2012). Increase in light enhance microalgal CO₂ sequestering in Chlorella vulgaris (Gonçalves et al. 2014), Alphanothece microscopica (Jacob-Lopes et al. 2009), and Chlorella PY-ZU1 (Cheng et al. 2013a). All mutagenesis studies of microalgae reported increase in CO₂ tolerance and fixation (Lee et al. 2002, Li et al. 2011, 2015, Kao et al. 2012b, Cheng et al. 2013b). Microalgae have shown promising potential for reduction of CO₂ in flue gas (Borkenstein et al. 2011, Chiu et al. 2011, Li et al. 2011, Cheng et al. 2014, 2015) and for simultaneously production of neutral lipid. Culture system can be combined wastewater treatment and make use impure CO₂ gas (Chinnasamy et al. 2010, Li et al. 2011). Several reports have highlighted the advantages (in terms of higher biomass yield and rate of pollutant removal) of coupled process of wastewater treatment with CO₂ from waste gases including flue gas (Kumar et al. 2011, Van Den Hende et al. 2011, Prajapati et al. 2013a). Moreover, CO₂ fraction of the biogas can also be used as carbon source for microalgal culturing at large scale (Kao et al. 2012a, Serejo et al. 2015).

Introducing abundant nutrients into biochemical cycles through agricultural practices, urbanization, and industrialization resulted in nutrient-enrich-water-bodies and caused dissolved oxygen depletion and eutrophication. Wastewater treatment can be achieved when it is used for culturing of microalgae by utilizing the nutrients.

Cyanides are toxic compounds containing carbon-nitrogen radicals (CN⁻) (e.g., hydrogen cyanide, sodium cyanide, potassium cyanide, and thiocyanates) and originate from manufacturing companies such as metal finishing, gold ore processing, plastic manufacturing, food processing, and steel production (Knowles and Bunch 1986, Gould et al. 2012). Biological methods, including use of microalgae, for removal of tightly bonded cyanide compounds from wastewater is cost effective and more efficient compared to chemical and physical counterparts (Gurbuz et al. 2009). Ability to withstand and / or utilize cyanide in wastewater varies among different algae species. Although growth of microalgae could be initially retarded by cyanide, some microalgae species acclimatized quickly. Algae is capable of withstanding higher (up to 400 mg L⁻¹) cyanide concentration (Gurbuz et al. 2002), an advantage over bacteria since bacteria that can only withstand maximum of 300 mg L⁻¹ (Adams et al. 2001). Currently, there is no report on molecular study of metabolism of cyanide in microalgae. Presumably, similar to other microorganisms, microalgae utilized cyanide through series of enzymatic reactions for bioconversion into simple organic or inorganic molecules. Vanelslander et al. (2011) reported production of a diverse mixture of iodinated and brominated metabolites including bromine cyanide and iodine cyanide by diatom Nitzschia cf pellucida. Numerous enzymes that are responsible for metabolism of cyanide compounds have been identified in plants, fungi, and bacteria. Hydrolytic pathway involves cyanide hydratase, nitrile hydratase, thiocyanate hydrolase, nitrilase, and cyanidase; oxidative pathway involves cyanide monooxygenase, cyanide cyanase, and cyanide dioxygenase; reductive pathway involves enzyme nitrogenase; substitution / transfer pathway involves mercaptopyruvate sulfurtransferase and rhodanese while syntheses pathway involves β-cyanoalanine synthase, and γ-cyano-α-aminobutyric acid synthase (Gupta et al. 2010, Gould et al. 2012). However, none of the aforementioned enzymes have been reportedly present in microalgae. Besides, microalgae have been shown to metabolize cyanide from wastewater. Microalgae assimilated cyanides compounds as source of carbon or nitrogen (Gupta et al. 2010). Additional work is needed to investigate the cyanide metabolic pathway in microalgae.

Utilization of pesticides is common agricultural practice to increase food production, but when overused, pesticides become harmful to soil, aquatic ecosystem, and human health (Zhang et al. 2011). Similarly, residue of pesticides on plant materials, soils, water bodies found their ways in to animals and human systems (Kannan et al. 1999). Microalgal treatment of water bodies has been identified as a very suitable approach for bioremediation of pesticides (Table 2).

Fresh water microalgae have exhibited different efficiencies for pesticides removal from wastewater (Dosnon-Olette et al. 2010). Fluorxypyr and isoproturon were rapidly accumulated into the microalgae C. reinhardtii cell matrix (Zhang et al. 2011). Studies have equally shown possibility of microalgae to biodegrade some pesticides. Microalgae exhibited different level of toxicological effects with respect to different pesticides. Most of the reports showed induction of oxidative responses by the microalgae in the presence of high concentration of pesticides. In addition, cell morphology was altered and cell photosynthetic apparatus were disrupted. The following toxicology reactions: decrease in soluble protein and total antioxidant content, increase in superoxide dismutase and peroxidase activity, reduction of transcription of photosynthetic genes and one energy gene and cellular structural damage (Shen et al. 2014). Esperanza et al. (2015) showed that C. reinhardtii, subjected to sublethal concentration of atrazine for 3 and 24 h, experienced changed in gene implicated in amino acid catabolism and respiratory cellular process. It was suggested that, photosynthesis was inhibited and the cell was forced to seek heterotrophic metabolism in order to survive. Ability to withstand certain concentration of specific pesticides varies among different species of microalgae. Microalgae are not only capable of bioaccumulation of pesticides, they can also biodegrade them when pesticides are presented in sublethal concentration. However, microalgal metabolic pathway for bioremediation of pesticides is still an area to be explored in future researches.

Endocrine disruptors are a group of compounds with estrogenic activities. They have been found in wastewaters, and also impacted several health issues in living organisms (Hom-Diaz et al. 2015). Endocrine disruptors causes morphological alterations, fertility reduction, interference in sex differentiation, enhancing growth of MCF-7 human breast cancer cell and mutagenic action on RSa human cells (Gattullo et al. 2012). Many studies on bioremediation of wastewater containing endocrine disruptors using microalgae have been conducted (Nadal et al. 2006, Della Greca et al. 2008, Li et al. 2009, Shi et al. 2010, Gattullo et al. 2012, Zhou et al. 2013, Hom-Diaz et al. 2015). Microalgae can tolerated high concentration of endocrine disruptors, therefore, they are suitable for bioremediation of endocrine disruptors (Perron and Juneau 2011). Abargues et al. (2013) showed that aerated system was more efficient for removal of micropollutants while unaerated system was only suitable for removal of 4-n-nonylphenol. In another study, Chlorella fisca was able to remove most of bisphenol A in presence of light. Almost all the bisphenol A was removed under continuous light, about 82% of bisphenol removed under light : dark (8 : 16) condition while 42% of bisphenol A was removed under dark condition (Hirooka et al. 2005).

The possible processes for bioremediation of endocrine disruptors in water are sorption, biodegradation, and photolytic degradation (Shi et al. 2010). Rate of bioremediation of endocrine disruptors, in algae system, varies among different algae species and inversely proportional to molecular weight of the endocrine disruptors (Ji et al. 2014a, Hom-Diaz et al. 2015). More studies on strain selection is required in order to identify microalgae with high bioremediation properties against endocrine disruptors. Likewise, other conditions (temperature, treatment time, presence of light and concentration, and type of endocrine disruptors) that affect bioremediation of endocrine disruptors need attention in future researches.

Bioremmediation has been identified as most suitable method to combat hydrocarbon contamination of soil and water as a result of industrial discharges (Subashchandrabose et al. 2013). Microalgae, as a member of microbial community, plays significant role toward bioremediation of toxic compounds including hydrocarbon. Microalgae has shown ability to produce enzymes capable of degrading hydrocarbon (Chekroun et al. 2014). Earlier studies on microalgal remediation of hydrocarbon polluted environment have proven that several species of microalgae are capable of utilizing hydrocarbons as carbon source (Semple et al. 1999). The most popular hydrocarbon degrading microalgae is Prototheca zopfii and has been investigated in cell free and immobilized systems (Suzuki and Yamaya 2005, Ueno et al. 2006, 2007, de-Bashan and Bashan 2010). Immobilization of microalgae reduced lag growth phase associated with biodegradation of n-alkanes. The immobilized thermotolerant strain selectively degraded aliphatic hydrocarbon in the mixed hydrocarbon substrate unlike the nonthermotolerant strain (Ueno et al. 2006). Microalgae have different affinity for degradation of n-alkanes and polycyclic aromatic hydrocarbons (Gamila and Ibrahim 2004). Scenedesmus obliquus showed greater affinity toward degradation of polycyclic hydrocarbons than n-alkanes, unlike Nitzschia linearis that exhibited higher preference for degradation of n-alkanes than polycyclic hydrocarbons. Also, seven microalgae isolated from Nile river exhibited different potentials when tested for bioremediation of crude oil (Ibrahim and Gamila 2004). Tang et al. (2010) and Hong et al. (2008) showed that degradation and accumulation abilities of Nitzschia sp. were higher than those of Skeletonema costatum and that it is easier to degrade fluoranthene compared to phenanthrene.

Microalgae has been reported to play important role, but not directly responsible, in microbial consortia for degradation of light extractable petroleum hydrocarbons in petroleum-contaminated water (Jacques and McMartin 2009). It is possible that produced oxygen by microalgae during photosynthesis process is easily utilized by bacteria (Muñoz and Guieysse 2006). Several microalgal-bacterial consortia have also been studied for bioremediation of hydrocarbons. Microalgal-bacterial consortium containing S. obliquus showed high degradation efficiency against aliphatic and aromatic hydrocarbons of crude oil (Tang et al. 2010). Another report showed that microalgal-bacterial consortium (Chlorella sorokiniana and Pseudomonas migulae) biodegraded between 200 to 500 mg L⁻¹ of phananthrene under photosynthetic conditions without external supply of oxygen (Muñoz et al. 2003). Additional studies are needed to explore metabolic pathway that is responsible for bioaccumulation and biodegration of hydrocarbon in microalgae and microalgal-bacterial consortia.