**2. Microalgae growth in wastewaters**

The cultivation of microalgae in wastewater has long been recognized as a viable option for sustainable biomass production and wastewater treatment [18–21]. The main nutritional requirements for microalgae growth include nitrogen, phosphorus, and micronutrients such as iron, magnesium, and calcium, which are present in wastewater. Recent developments in microalgal research have demonstrated that microalgae have the required metabolic potential to effectively reduce high concentrations of nutrients such as carbon, phosphorous, and nitrogen present in different wastewater streams [21]. Some species of microalgae have the ability to take up other pollutants, such as heavy metals and harmful chemicals [20]. Therefore, microalgae can be used to serve a dual purpose for the treatment of wastewater as well as generating biomass for various applications because microalgae are rich in carbohydrates, proteins, and lipids.

Various wastewater streams including municipal, industrial, agricultural wastewater, as well as primary and secondary effluent, centrate, and anaerobic digestion effluent were exploited as suitable nutrient media for microalgae cultivation. Each wastewater stream has its own characteristics and challenges such as nutrient variability and the presence of potential inhibitors that could impact microalgal growth. Recently, many investigations have been developed to overcome challenges such as low nutrients, high turbidity, bacterial contamination, and specific toxic materials associated with different wastewaters.

The types of wastewater utilized for algae cultivation also affect the scope of biomass for various applications [21].

An alternative for recovering energy from microalgae is based on the application of anaerobic digestion processes [35]. In such processes, all organic matters (proteins, carbohydrates, and lipids) present in microalgae biomass would be converted into methane and carbon dioxide (biogas). Several advantages are recognized when energy production from whole microalgae through biogas generation is considered: biogas production involves high-energy yields; biogas production would not require microalgae biomass drying (it involves wet fermentation); biogas can be used to produce heat and electricity through co-generation; microalgae cultures can be used for biogas upgrading (i.e. CO<sup>2</sup> biosequestration); and so on. However, some microalgae have a very low C/N ratio, which hinders and inhibits a further anaerobic digestion. Ammonia toxicity and recalcitrant cell walls are commonly cited causes of the low-methane yields found in the anaerobic digestion of some microalgae [36]. Moreover, anaerobic co-digestion of microalgae with other types of biomass such as solid and liquid wastes is quite feasible [35]. The benefits of co-digestion lie in balancing the C/N ratio in the co-substrate mixture, as well as macro and micronutrients, pH, inhibitor/toxic compounds, and dry matter [37].

The main phyla (and species) of microalgae that are being used for biogas production through anaerobic digestion and co-digestion processes are as follows [20, 21, 38]:


#### **2.1. Chlorophytes**

digestion of two or more substrates, and it is a proven approach to overcome the drawbacks of single digestion [32]. Mata-Alvarez et al. [33] in the year 2000 already wrote: "The use of a co-substrate, in most cases improves biogas yield due to positive synergisms established in the digestion medium and the supply of missing nutrients by the co-substrates." Co-digestion has several advantages as follows: adjusting the C/N ratio, improving the nutrients, and diluting the inhibitor compounds [34]. The co-digestion of microalgae with high-carbon biomass leads to a better balanced substrate for anaerobic digestion [12, 13, 27]. Nevertheless, there are some problems that must be solved, such as the breakage of the thick cellular walls in some microalgae and cyanobacteria. Prospective methods could be different kinds of pretreatments

Nonetheless, due to the high variety of microalgae and cyanobacteria and the wide range of different uses, it is not clear yet what the most effective process for biofuel production is. Although to this respect, some authors suggest that the direct use of microalgae or cyanobacteria in an anaerobic co-digestion process is the best choice, while other researchers propose that the best choice is to produce biofuel as a first step followed by an anaerobic digestion of the residual by-products [1]. This chapter aims at providing a current perspective of microalgae exploitation as biomass in anaerobic digestion and co-digestion processes and shows the

The cultivation of microalgae in wastewater has long been recognized as a viable option for sustainable biomass production and wastewater treatment [18–21]. The main nutritional requirements for microalgae growth include nitrogen, phosphorus, and micronutrients such as iron, magnesium, and calcium, which are present in wastewater. Recent developments in microalgal research have demonstrated that microalgae have the required metabolic potential to effectively reduce high concentrations of nutrients such as carbon, phosphorous, and nitrogen present in different wastewater streams [21]. Some species of microalgae have the ability to take up other pollutants, such as heavy metals and harmful chemicals [20]. Therefore, microalgae can be used to serve a dual purpose for the treatment of wastewater as well as generating biomass for various applications because microalgae are rich in carbohydrates,

Various wastewater streams including municipal, industrial, agricultural wastewater, as well as primary and secondary effluent, centrate, and anaerobic digestion effluent were exploited as suitable nutrient media for microalgae cultivation. Each wastewater stream has its own characteristics and challenges such as nutrient variability and the presence of potential inhibitors that could impact microalgal growth. Recently, many investigations have been developed to overcome challenges such as low nutrients, high turbidity, bacterial contamination,

The types of wastewater utilized for algae cultivation also affect the scope of biomass for vari-

before anaerobic digestion in some particular cases.

**2. Microalgae growth in wastewaters**

proteins, and lipids.

62 Microalgal Biotechnology

ous applications [21].

advantages of their growth in wastewater and anaerobic digestates.

and specific toxic materials associated with different wastewaters.

#### *2.1.1. Chlorella genus*

The growth of the green algae *Chlorella* sp. in wastewater after primary settling of a local municipal wastewater treatment plant was evaluated by Wang et al. [39]. They observed a growth rate of 0.429 d−1 with excellent removal of ammonium (NH<sup>4</sup> + -N) (74.7%), P (90.6%), and COD (56.5%). These authors also investigated the growth of *Chlorella* sp. using different phases (raw, secondary, and centrate) and demonstrated that the growth rate of microalgae and nutrient removal efficiencies was proportional to the nutrient concentration of the wastewater selected for its cultivation with the highest growth in centrate followed by raw wastewater. Osundeko and Pittman [40] reported a high-sodium concentration of 400 mg/L in sludge liquor/centrate, which can be toxic to freshwater microalgal species, though some *Chlorella* sp. are tolerant to salinity. More recently, Lu et al. [41] evaluated the biomass productivity and nutrient removal capacity of *Chlorella* sp. in raw dairy wastewater using both indoor bench-scale and outdoor pilot-scale photobioreactors. Results from this study have shown a higher biomass productivity of 260 mg/(L·d) and high nutrient (N and P) removal (83.3 and 38.3 mg/(L·d), respectively) in indoor bench-scale cultures when compared to outdoor pilot-scale cultures with biomass of 110 mg/(L·d) and nutrient removal of 41.3 mg/(L·d) for N and 6.5 mg/(L·d) for P. These differences could have resulted due to the uncontrolled environmental and operational factors that might have affected the microalgae growth during outdoor cultivation.

digester; the microalgal biomass was then anaerobically digested to produce methane. In a later study, with hydraulic retention time (HRT) of 28 days, 51% COD removal and methane production of 240 mL/g VSS were achieved. The use of microalgae as a feedstock for bioethanol production is considered to be a sustainable approach to bioethanol production. Microalgal species such as *Chlorella* store energy in the form of starch [48]. The starch accumulated in the microalgae can be easily hydrolyzed to glucose using chemical or enzymatic method. The sugar produced can be subsequently fermented to ethanol. Ho et al. [48] investigated the potential of *C. vulgaris* PS-E as the bioethanol feedstock. This species contains 51% of carbohydrates, which were hydrolyzed through an enzymatic process to give a glucose yield of 0.461 g glucose/g dry biomass. The ethanol yield obtained in their study was 11.7 g/L. *C. vulgaris* was also reported to be a successful bioremediation agent of palm oil mill effluent (POME), with reductions of ammonia-nitrogen, phosphorus, COD, and biochemical oxygen demand (BOD) of 61, 84, 50.5, and 61.6%, respectively [49]. Bich et al. [50] reported that *C. vulgaris* was used in the treatment of rubber latex concentrate processing wastewater and that this microalga reduced the COD and total Kjeldahl nitrogen (TKN) by 93.4 and 79.3%, respectively. Another study carried out by Nordin et al. [51] used high-rate algal ponds (HRAPs) to treat rubber effluent from an anaerobic digester, and the reductions in COD, BOD, NH<sup>3</sup>

The Influence of Microalgae Addition as Co-Substrate in Anaerobic Digestion Processes

http://dx.doi.org/10.5772/intechopen.75914

and phosphorous reached 69.1, 87.4, 62.2, and 21.3%, respectively. In the HRAP, *Chlorella* was

Moderately polluted textile wastewater was previously reported to be treated using the microalga *C. vulgaris*, with color and COD reductions of up to 69.9 and 75.7%, respectively [52]. Another study found that this species could degrade 63–69% mono-azo dyes into simple aromatic compounds [53]. Lim et al. [54] investigated the treatment of textile wastewater using 10 different strains of microalgae and found that *C. vulgaris* was able to remove color from the wastewater. When cultured in a HRAP, color removal reached 50% along with high

Two wild-type green algae such as *Micractinium* sp. and *Chlorella* sp. can also be grown in high-nitrogen wastewater (mixture of sludge centrate and primary effluent wastewater). The extraction and analysis of extracellular polymeric substances (EPSs) in both algal species during cultivation showed that *Micractinium* generated a higher amount of EPS proteins than *Chlorella* [27]. This fact affects the anaerobic biodegradability and methane yield when these

Food wastewater (FW), rich in nutrients including N, P, Ca, Fe, Al, and total organic carbon (TOC), was also effectively used for microalgal cultivation [9]. The effect of FW supplementation on the biomass and lipid productivity of *Scenedesmus obliquus* cultivated in Bold's Basal Medium (BBM) was recently investigated by Ji et al. [9]. They reported a substantial increase in growth and lipid productivity with supplementation of 1% FW to BBM. Furthermore, the fatty acid methyl ester (FAME) analysis revealed that the palmitic and oleic acid contents increased by up to 8% with the addition of FW. They also noted that FW promoted algal autoflocculation due to the formation of inorganic precipitates at an alkaline pH [9]. Similarly, the

the predominant genus [51].

reductions in COD, PO<sup>4</sup>

*2.1.2. Scenedesmus genus*

3−-P, and NH<sup>4</sup>

+ -N [54].

algae are anaerobically co-digested with waste-activated sludge (WAS).


65

Nutrient limitation is one of the key challenges for microalgal cultivation in secondary/tertiary wastewater. The supplementation of nutrients is proposed as an alternative method to overcome the nutrient limitations in wastewater. In this sense, Cabanelas et al. [42] identified the potential of coupling a wastewater treatment plant effluent with glycerol for supporting the mixotrophic production of *Chlorella vulgaris* and *Belippo terribilis*. The cultivation of *C. vulgaris* in mixotrophic mode was also studied in a mixture of primary and secondary wastewaters with different ratios (25, 50, and 75 vol.% of the primary wastewater). It was observed that using 25% of the primary wastewater and 75% of secondary wastewater resulted in 100% of COD removal, 100% of ammonium removal, and 82% of nitrate removal [43].

Recently, Ansari et al. [44] studied the cultivation of *Chlorella sorokiniana* in aquaculture wastewater with sodium nitrate supplementation and observed comparable biomass yields to the synthetic medium. In their study, they also observed high ammonia, nitrate, COD, and phosphate removal and proposed that treated water can be redirected toward aquaculture. The biomass obtained in this study showed sufficient lipid, carbohydrate, and protein concentrations to be used as feed supplement. Ramanna et al. [45] supplemented 1.5 g/L urea as a cheap N source for the cultivation of *C. sorokiniana* and achieved a biomass production of 0.218 g/L. A supplementation strategy can yield high-biomass productivities; however, it depends on the nutrient composition of the wastewater used and the requirements of the selected microalgal strain.

For the realization of microalgal CO<sup>2</sup> capture and utilization, the selection of microalgal species tolerant to CO2 from various environments and the characterization of growth influencing environmental factors are required [46]. The proper selection of species and optimized cultivation conditions, i.e., light intensity, temperature, nutrient availability, and pH, can maximize CO<sup>2</sup> sequestration*. Chlorella* sp. has been widely reported to possess good carbon sequestration potential. Previous studies have obtained hydrocarbons from microbial lipids for their conversion into sustainable fuels as a substitute for fossil hydrocarbons. Furthermore, microalgae have significant applications in the production of valuable materials in the food and pharmaceutical industries, resulting in a high value-added process in the biosequestration of CO2 [46].

Microalgae with a lipid content of lower than 40% of their dry weight make the anaerobic digestion route more feasible than biodiesel in terms of energy recovery. Ras et al. [47] proposed coupling the process of microalgal biomass production and anaerobic digestion. In this process, *C. vulgaris* was cultivated using the nutrient-rich digestate from an anaerobic digester; the microalgal biomass was then anaerobically digested to produce methane. In a later study, with hydraulic retention time (HRT) of 28 days, 51% COD removal and methane production of 240 mL/g VSS were achieved. The use of microalgae as a feedstock for bioethanol production is considered to be a sustainable approach to bioethanol production. Microalgal species such as *Chlorella* store energy in the form of starch [48]. The starch accumulated in the microalgae can be easily hydrolyzed to glucose using chemical or enzymatic method. The sugar produced can be subsequently fermented to ethanol. Ho et al. [48] investigated the potential of *C. vulgaris* PS-E as the bioethanol feedstock. This species contains 51% of carbohydrates, which were hydrolyzed through an enzymatic process to give a glucose yield of 0.461 g glucose/g dry biomass. The ethanol yield obtained in their study was 11.7 g/L.

*C. vulgaris* was also reported to be a successful bioremediation agent of palm oil mill effluent (POME), with reductions of ammonia-nitrogen, phosphorus, COD, and biochemical oxygen demand (BOD) of 61, 84, 50.5, and 61.6%, respectively [49]. Bich et al. [50] reported that *C. vulgaris* was used in the treatment of rubber latex concentrate processing wastewater and that this microalga reduced the COD and total Kjeldahl nitrogen (TKN) by 93.4 and 79.3%, respectively. Another study carried out by Nordin et al. [51] used high-rate algal ponds (HRAPs) to treat rubber effluent from an anaerobic digester, and the reductions in COD, BOD, NH<sup>3</sup> -N, and phosphorous reached 69.1, 87.4, 62.2, and 21.3%, respectively. In the HRAP, *Chlorella* was the predominant genus [51].

Moderately polluted textile wastewater was previously reported to be treated using the microalga *C. vulgaris*, with color and COD reductions of up to 69.9 and 75.7%, respectively [52]. Another study found that this species could degrade 63–69% mono-azo dyes into simple aromatic compounds [53]. Lim et al. [54] investigated the treatment of textile wastewater using 10 different strains of microalgae and found that *C. vulgaris* was able to remove color from the wastewater. When cultured in a HRAP, color removal reached 50% along with high reductions in COD, PO<sup>4</sup> 3−-P, and NH<sup>4</sup> + -N [54].

Two wild-type green algae such as *Micractinium* sp. and *Chlorella* sp. can also be grown in high-nitrogen wastewater (mixture of sludge centrate and primary effluent wastewater). The extraction and analysis of extracellular polymeric substances (EPSs) in both algal species during cultivation showed that *Micractinium* generated a higher amount of EPS proteins than *Chlorella* [27]. This fact affects the anaerobic biodegradability and methane yield when these algae are anaerobically co-digested with waste-activated sludge (WAS).

#### *2.1.2. Scenedesmus genus*

*Chlorella* sp. are tolerant to salinity. More recently, Lu et al. [41] evaluated the biomass productivity and nutrient removal capacity of *Chlorella* sp. in raw dairy wastewater using both indoor bench-scale and outdoor pilot-scale photobioreactors. Results from this study have shown a higher biomass productivity of 260 mg/(L·d) and high nutrient (N and P) removal (83.3 and 38.3 mg/(L·d), respectively) in indoor bench-scale cultures when compared to outdoor pilot-scale cultures with biomass of 110 mg/(L·d) and nutrient removal of 41.3 mg/(L·d) for N and 6.5 mg/(L·d) for P. These differences could have resulted due to the uncontrolled environmental and operational factors that might have affected the microalgae growth during

Nutrient limitation is one of the key challenges for microalgal cultivation in secondary/tertiary wastewater. The supplementation of nutrients is proposed as an alternative method to overcome the nutrient limitations in wastewater. In this sense, Cabanelas et al. [42] identified the potential of coupling a wastewater treatment plant effluent with glycerol for supporting the mixotrophic production of *Chlorella vulgaris* and *Belippo terribilis*. The cultivation of *C. vulgaris* in mixotrophic mode was also studied in a mixture of primary and secondary wastewaters with different ratios (25, 50, and 75 vol.% of the primary wastewater). It was observed that using 25% of the primary wastewater and 75% of secondary wastewater resulted in 100%

Recently, Ansari et al. [44] studied the cultivation of *Chlorella sorokiniana* in aquaculture wastewater with sodium nitrate supplementation and observed comparable biomass yields to the synthetic medium. In their study, they also observed high ammonia, nitrate, COD, and phosphate removal and proposed that treated water can be redirected toward aquaculture. The biomass obtained in this study showed sufficient lipid, carbohydrate, and protein concentrations to be used as feed supplement. Ramanna et al. [45] supplemented 1.5 g/L urea as a cheap N source for the cultivation of *C. sorokiniana* and achieved a biomass production of 0.218 g/L. A supplementation strategy can yield high-biomass productivities; however, it depends on the nutrient composition of the wastewater used and the requirements of the

ing environmental factors are required [46]. The proper selection of species and optimized cultivation conditions, i.e., light intensity, temperature, nutrient availability, and pH, can

sequestration potential. Previous studies have obtained hydrocarbons from microbial lipids for their conversion into sustainable fuels as a substitute for fossil hydrocarbons. Furthermore, microalgae have significant applications in the production of valuable materials in the food and pharmaceutical industries, resulting in a high value-added process in the biosequestra-

Microalgae with a lipid content of lower than 40% of their dry weight make the anaerobic digestion route more feasible than biodiesel in terms of energy recovery. Ras et al. [47] proposed coupling the process of microalgal biomass production and anaerobic digestion. In this process, *C. vulgaris* was cultivated using the nutrient-rich digestate from an anaerobic

capture and utilization, the selection of microalgal spe-

from various environments and the characterization of growth influenc-

sequestration*. Chlorella* sp. has been widely reported to possess good carbon

of COD removal, 100% of ammonium removal, and 82% of nitrate removal [43].

outdoor cultivation.

64 Microalgal Biotechnology

selected microalgal strain.

[46].

cies tolerant to CO2

maximize CO<sup>2</sup>

tion of CO2

For the realization of microalgal CO<sup>2</sup>

Food wastewater (FW), rich in nutrients including N, P, Ca, Fe, Al, and total organic carbon (TOC), was also effectively used for microalgal cultivation [9]. The effect of FW supplementation on the biomass and lipid productivity of *Scenedesmus obliquus* cultivated in Bold's Basal Medium (BBM) was recently investigated by Ji et al. [9]. They reported a substantial increase in growth and lipid productivity with supplementation of 1% FW to BBM. Furthermore, the fatty acid methyl ester (FAME) analysis revealed that the palmitic and oleic acid contents increased by up to 8% with the addition of FW. They also noted that FW promoted algal autoflocculation due to the formation of inorganic precipitates at an alkaline pH [9]. Similarly, the biomass, lipid productivity, and nutrient removal efficiency of *S. obliquus* cultivated under mixotrophic conditions in municipal wastewater were reportedly enhanced when supplemented with FW and flue gas CO<sup>2</sup> [55].

through bioremediation [62]. Individual contaminants (As, Cd, Cr, Co, Cu, Pb, Ni, Hg, Se, and Zn) at various concentrations ranging from a low concentration (1X) to higher concentrations (10X and 40X) found in contaminated systems (mine tailings, wastewater treatment plants, produced water) were introduced into growth media. Biological growth experimentation was performed in triplicate at the various contaminant concentrations and at three different light intensities. Results showed that baseline concentrations of each contaminant slightly decreased biomass growth between 89 and 99% of the control with the exception of Ni, which dramatically reduced growth. Increased contaminant concentrations resulted in progressively lower growth rates for all the contaminants tested. Lipid analysis showed that most baseline contaminant concentrations slightly decreased or had minimal effects on lipid content at all light levels. Trace contaminant analysis on the biomass showed that Cd, Co, Cu, Pb, and Zn were sorbed by the microalgae with minimal contaminants remaining in the growth media, which illustrated the effectiveness of microalgae to bioremediate these contaminants when levels are sufficiently low and to not detrimentally impact productivity. The

The Influence of Microalgae Addition as Co-Substrate in Anaerobic Digestion Processes

http://dx.doi.org/10.5772/intechopen.75914

67

microalgae biomass was less efficient in the sorption of As, Cr, Ni, and Se [62].

minus nitrogen and phosphorus [63].

*2.1.5. Botryococcus braunii*

with CO2

was achieved [64].

Another study revealed that metal levels in municipal wastewaters were unlikely to inhibit algal growth and lipid production at least by metals, which are tolerant to microalgae like *N. salina*. Cells grew without inhibition in treated municipal wastewater or centrate derived from wastewater treatment with the addition of up to 75% v/v in their normal growth medium

*B. braunii* is a microalga, which is regarded as a potential source of renewable fuel because of its ability to produce large amounts of lipids that can be converted into biodiesel. Agroindustrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. Two strategies for the low-cost production of *B. braunii* biomass with high-lipid content were performed: (i) mixotrophic cultivation using molasses, a cheap by-product from the sugar cane plant as a carbon source, and (ii) photoautotrophic cultivation using nitrate-rich wastewater supplemented

high amount of biomass at 3.05 g/L with a high-lipid content of 36.9%. The photoautotrophic

2.26 g/L and a lipid content of 30.3%. The benefits of this photoautotrophic cultivation are that this cultivation would help to reduce the accumulation of atmospheric carbon dioxide and more than 90% of the nitrate could be removed from the wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with the semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g/L with a comparable lipid content of 32.2%

To understand the potential of using swine lagoon wastewater to cultivate *B. braunii* for biofuel production, the growth characteristics of *B. braunii* 765 cultivated in aerated swine lagoon wastewater (ASLW) without sterilization and pH adjustment were investigated. The results showed that the alga strain could maintain a competitive advantage over the 26-day cultivation. The highest dry biomass of alga grown in ASLW was 0.94 mg/L at Day 24, which was 1.73 times that

cultivation in nitrate-rich wastewater supplemented with 2.0% CO<sup>2</sup>

as a carbon source. Mixotrophic cultivation added with 15 g/L molasses produced a

produced a biomass of

Shanab et al. [56] demonstrated that out of three fresh water microalgal isolates selected for heavy metal tolerance studies, *Scenedesmus quadricauda* showed tolerance to heavy metals such as Hg2+, Pb2+, and Cd2+ in up to 100 mg/L concentrations. Research on the applications of immobilized microalgal cells indicated that immobilized algal cells are more tolerant to heavy metal stress when compared to free living cells [56].

*Scenedesmus* sp. has also been widely reported with good carbon sequestration potential [57]. These studies obtained hydrocarbons from microalgal lipids for their conversion into sustainable biofuels as a substitute for fossil hydrocarbons. Furthermore, microalgae have significant applications in the production of valuable materials in the food and pharmaceutical industries, producing a high value-added process in the biosequestration of CO<sup>2</sup> [57].

Similar to bioconversion, some microalgae can also carry out the biosorption of textile wastewater. For instance, *S. quadricauda* has been successfully employed as biosorbent to remove remazol brilliant blue R (RBBR) [58, 59].

In a very recent study, microalgae digestate and secondary effluent were used to grow *Scenedesmus* sp. in a tertiary treatment using a 30 L closed photobioreactor for cultivation. The microalgae biomass, composed of *Scenedesmus* sp., reached and maintained a concentration of 1.1 g TSS/L during 30 days [22]. A complete removal of N-NH<sup>4</sup> + and P-PO<sup>4</sup> 3− and high nitrate and organic matter removals were achieved (58% N-NO<sup>3</sup> − and 70% COD) with 8 days of HRT [22].

#### *2.1.3. Dunaliella salina*

A very recent study assessed the feasibility of the cultivation of *D. salina* in controlled environment tertiary-treated municipal wastewater [60]. *D. salina* was selected for its high β-carotene generation capacity and for being a halophilic species to protect our fresh water resources further through wastewater remediation. Nutrient analyses indicated that *D. salina* can significantly remove nitrate, ammonia, and phosphorus from municipal wastewater in the range of 45–88%. Among all combinations studied, optimal algal growth was observed at 30 ppt salinity level, with a 75% wastewater concentration (3:1 ratio of wastewater and saline water mixture, which is the growth medium). These findings concluded that *D. salina* has great capacity for nutrient uptake while providing high-value bioproducts [60].

Another study assessed the production rates of some native microalgae growing in media supplemented with algal digestate, urban wastewater, or digested sludge. Very low production rates, or no growth, were measured when microalgae isolated from high-salinity waters (*D. salina*) were used, suggesting that populations well adapted to extreme environmental conditions are not suitable candidates for growing in wastewater or anaerobic digestate [61].

#### *2.1.4. Nannochloropsis salina*

The potential for *N. salina* to be integrated with contaminated water sources was assessed for the concurrent production of a biofuel feedstock while providing an environmental service through bioremediation [62]. Individual contaminants (As, Cd, Cr, Co, Cu, Pb, Ni, Hg, Se, and Zn) at various concentrations ranging from a low concentration (1X) to higher concentrations (10X and 40X) found in contaminated systems (mine tailings, wastewater treatment plants, produced water) were introduced into growth media. Biological growth experimentation was performed in triplicate at the various contaminant concentrations and at three different light intensities. Results showed that baseline concentrations of each contaminant slightly decreased biomass growth between 89 and 99% of the control with the exception of Ni, which dramatically reduced growth. Increased contaminant concentrations resulted in progressively lower growth rates for all the contaminants tested. Lipid analysis showed that most baseline contaminant concentrations slightly decreased or had minimal effects on lipid content at all light levels. Trace contaminant analysis on the biomass showed that Cd, Co, Cu, Pb, and Zn were sorbed by the microalgae with minimal contaminants remaining in the growth media, which illustrated the effectiveness of microalgae to bioremediate these contaminants when levels are sufficiently low and to not detrimentally impact productivity. The microalgae biomass was less efficient in the sorption of As, Cr, Ni, and Se [62].

Another study revealed that metal levels in municipal wastewaters were unlikely to inhibit algal growth and lipid production at least by metals, which are tolerant to microalgae like *N. salina*. Cells grew without inhibition in treated municipal wastewater or centrate derived from wastewater treatment with the addition of up to 75% v/v in their normal growth medium minus nitrogen and phosphorus [63].

#### *2.1.5. Botryococcus braunii*

biomass, lipid productivity, and nutrient removal efficiency of *S. obliquus* cultivated under mixotrophic conditions in municipal wastewater were reportedly enhanced when supple-

Shanab et al. [56] demonstrated that out of three fresh water microalgal isolates selected for heavy metal tolerance studies, *Scenedesmus quadricauda* showed tolerance to heavy metals such as Hg2+, Pb2+, and Cd2+ in up to 100 mg/L concentrations. Research on the applications of immobilized microalgal cells indicated that immobilized algal cells are more tolerant to heavy

*Scenedesmus* sp. has also been widely reported with good carbon sequestration potential [57]. These studies obtained hydrocarbons from microalgal lipids for their conversion into sustainable biofuels as a substitute for fossil hydrocarbons. Furthermore, microalgae have significant applications in the production of valuable materials in the food and pharmaceutical indus-

Similar to bioconversion, some microalgae can also carry out the biosorption of textile wastewater. For instance, *S. quadricauda* has been successfully employed as biosorbent to remove

In a very recent study, microalgae digestate and secondary effluent were used to grow *Scenedesmus* sp. in a tertiary treatment using a 30 L closed photobioreactor for cultivation. The microalgae biomass, composed of *Scenedesmus* sp., reached and maintained a concentration of

A very recent study assessed the feasibility of the cultivation of *D. salina* in controlled environment tertiary-treated municipal wastewater [60]. *D. salina* was selected for its high β-carotene generation capacity and for being a halophilic species to protect our fresh water resources further through wastewater remediation. Nutrient analyses indicated that *D. salina* can significantly remove nitrate, ammonia, and phosphorus from municipal wastewater in the range of 45–88%. Among all combinations studied, optimal algal growth was observed at 30 ppt salinity level, with a 75% wastewater concentration (3:1 ratio of wastewater and saline water mixture, which is the growth medium). These findings concluded that *D. salina* has great

Another study assessed the production rates of some native microalgae growing in media supplemented with algal digestate, urban wastewater, or digested sludge. Very low production rates, or no growth, were measured when microalgae isolated from high-salinity waters (*D. salina*) were used, suggesting that populations well adapted to extreme environmental conditions are not suitable candidates for growing in wastewater or anaerobic digestate [61].

The potential for *N. salina* to be integrated with contaminated water sources was assessed for the concurrent production of a biofuel feedstock while providing an environmental service

−

+

and P-PO<sup>4</sup>

and 70% COD) with 8 days of HRT [22].

[57].

3− and high nitrate and

[55].

tries, producing a high value-added process in the biosequestration of CO<sup>2</sup>

capacity for nutrient uptake while providing high-value bioproducts [60].

1.1 g TSS/L during 30 days [22]. A complete removal of N-NH<sup>4</sup>

organic matter removals were achieved (58% N-NO<sup>3</sup>

mented with FW and flue gas CO<sup>2</sup>

66 Microalgal Biotechnology

metal stress when compared to free living cells [56].

remazol brilliant blue R (RBBR) [58, 59].

*2.1.3. Dunaliella salina*

*2.1.4. Nannochloropsis salina*

*B. braunii* is a microalga, which is regarded as a potential source of renewable fuel because of its ability to produce large amounts of lipids that can be converted into biodiesel. Agroindustrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. Two strategies for the low-cost production of *B. braunii* biomass with high-lipid content were performed: (i) mixotrophic cultivation using molasses, a cheap by-product from the sugar cane plant as a carbon source, and (ii) photoautotrophic cultivation using nitrate-rich wastewater supplemented with CO2 as a carbon source. Mixotrophic cultivation added with 15 g/L molasses produced a high amount of biomass at 3.05 g/L with a high-lipid content of 36.9%. The photoautotrophic cultivation in nitrate-rich wastewater supplemented with 2.0% CO<sup>2</sup> produced a biomass of 2.26 g/L and a lipid content of 30.3%. The benefits of this photoautotrophic cultivation are that this cultivation would help to reduce the accumulation of atmospheric carbon dioxide and more than 90% of the nitrate could be removed from the wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with the semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g/L with a comparable lipid content of 32.2% was achieved [64].

To understand the potential of using swine lagoon wastewater to cultivate *B. braunii* for biofuel production, the growth characteristics of *B. braunii* 765 cultivated in aerated swine lagoon wastewater (ASLW) without sterilization and pH adjustment were investigated. The results showed that the alga strain could maintain a competitive advantage over the 26-day cultivation. The highest dry biomass of alga grown in ASLW was 0.94 mg/L at Day 24, which was 1.73 times that grown in a BG 11 medium, an artificial medium normally used for *B. braunii* cultivation. And the algal hydrocarbon content was 23.8%, which was more than twice that in the BG 11 medium. Additionally, after the 26-day cultivation period, about 40.8% of TN and 93.3% of TP in ASLW were removed, also indicating good environmental benefits of algal bioremediation [65].

cyanobacteria *A. platensis* cultivated in aquaculture wastewater as algal biofertilizer for the leafy vegetables Arugula (*Eruca sativa*), Bayam Red (*Amaranthus gangeticus*) and Pak Choy (*Brassica rapa* ssp. *chinensis*). In their study, *A. platensis* biomass showed lower amounts of NPK, while amounts of iron, magnesium, calcium, and zinc were found to be higher in algal

Microalgae are a rich source of proteins, pigments, and omega fatty acids and thus find application in human and animal feed production. *A. platensis* is one of the dominant species of microalgae used in the health food industry [69].The omega fatty acids from this microalga are used as human food and animal feed supplements. Phang et al. [70] found that the biomass composition of *Arthrospira* cultured in a high-rate algal pond for the treatment of sago starch processing wastewater can be used as high-quality animal feed, especially in the aquaculture industry. During the mentioned treatment of sago processing wastewater using *Spirulina*,

Zainal et al. [71] reported that *A. platensis* was able to treat wastewater containing heavy metals and removed manganese by 84.9%; chromium by 83.8%; arsenic by 71.4%; nickel by 61.9%;

Similar to bioconversion, microalgae could also carry out the biosorption of textile wastewater. For instance, *A. platensis* was used as a biosorbent to remove reactive red 120 (RR-120) from its aqueous solution. It achieved the maximum biosorption capacity of 482.2 mg/g

The performance of *O. tenuis* to remove nitrogen, phosphorus, and COD from secondary effluents of municipal domestic wastewater was investigated in batch experiments. *O. tenuis*

phosphorus and COD removal efficiencies of 82.9 and 92.6%, respectively, within 7 days at an

At the same time, *O. tenuis* showed its capacity to remove reactive dyes from textile wastewater. This species degraded azo dyes into simple aromatic amines and decolorized dye waste-

Maintaining the uni-algal system requires a super clean environment, which can be attained under laboratory conditions only. In the outdoor cultivation of microalgae, it is almost impossible to maintain a uni-algal system. If so, it requires a lot of expertise and skills. Moreover, the biomass productivity of the uni-algal system is limited because of suppressed metabolic activity during night time or dark periods. Alternatively, heterotrophic microalgae are used, which are less sensitive to photoperiods, grow fast, and return high-biomass yields. However,

autotrophic microalgae in the cultivation matrix. Therefore, the concept of a binary culture system arises [38]. Binary culture is considered superior to the uni-algal system in several

is produced during oxidative metabolism, which remains un-

can be further utilized by employing

had a biomass productivity of 150 mL/(L·d), a removal rate of NH<sup>4</sup>


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+


biomass when compared to chemical fertilizer (Triple Pro 15-15-15).

COD, PO<sup>4</sup>

3−-P, and NH<sup>4</sup>

*2.3.2. Oscillatoria tenuis*

water [59].

aeration rate of 1.0 L/min [73].

a significant amount of CO<sup>2</sup>

**2.4. Binary and mixed culture systems**

used and is released into the environment. This CO<sup>2</sup>

+

zinc by 55%; copper by 52.8%, and iron by 45.1%.

removing 97% RR-120 from the solution [72].

A study was conducted to evaluate the possibility of using wastewater from a soybean curd manufacturing plant as a growth promoter of *B. braunii* strain BOT-22. Soybean curd wastewater (SCW) was added to a AF-6 medium to set final concentrations at 0 (control), 1, 2, 5, and 10% (v/v). The growth and hydrocarbon production observed in the cultures with 1 and 2% SCW were significantly higher than that observed in the control. It was postulated that proteins and/or reducing sugars in SCW could enhance the growth [66].

#### *2.1.6. Micractinium genus*

The strain *Micractinium* sp. IC-76 was grown in municipal wastewater and showed a biomass productivity of 37.1 ± 4.1 mg/(L d) and a lipid content of 36.2 ± 0.1%, with a total content of saturated and monounsaturated fatty acids of 71.9%. The efficiency of nitrogen (N-NH<sup>4</sup> + ) and phosphorus (P-PO<sup>4</sup> 3−) removal was 96.4 ± 0.7 and 77.8 ± 5.6%, respectively. The strain *Micractinium* sp. IC-76 in the stationary phase of growth showed a significant difference in carbohydrate metabolism, especially sucrose concentration. High-lipid induction during cultivation in wastewater was also driven by changes in the biosynthesis of amino acids, fatty acids, and the tricarboxylic acid cycle [67].

*Micractinium* sp. Embrapa|LBA32 presented vigorous growth in a light-dependent manner in undiluted vinasse under non-axenic conditions. Microalgae strains presented higher biomass productivity in vinasse-based media when compared to standard BBM in cultures performed using 15 L airlift flat plate photobioreactors. Chemical composition analyses showed that proteins and carbohydrates comprise the major fractions of algal biomass. Glucose was the main monosaccharide detected, ranging from 46 to 76% of the total carbohydrate contents according to the culture media used [68].

#### **2.2. Haptophytes:** *Isochrysis galbana*

A recent study investigates the capacity of *I. galbana* in the bioremediation of aquaculture wastewater from a gray mullet *Mugil cephalus*. The experiment was conducted in batch conditions for 7 days using completely mixed bubble column photobioreactors. After 2 days, *I. galbana* removed 32 and 79% of dissolved inorganic nitrogen and dissolved inorganic phosphorus, respectively [10].

It has been also reported that *I. galbana* cultured in open ponds has fatty acids and a highprotein content, which are suitable for animal nutrition [20].

#### **2.3. Cyanobacteria**

#### *2.3.1. Arthrospira platensis*

Phosphorus can be recycled from wastewater through microalgal cultivation and provided to crop plants in the form of microalgal biofertilizers. Guldhe et al. [21] reported filamentous cyanobacteria *A. platensis* cultivated in aquaculture wastewater as algal biofertilizer for the leafy vegetables Arugula (*Eruca sativa*), Bayam Red (*Amaranthus gangeticus*) and Pak Choy (*Brassica rapa* ssp. *chinensis*). In their study, *A. platensis* biomass showed lower amounts of NPK, while amounts of iron, magnesium, calcium, and zinc were found to be higher in algal biomass when compared to chemical fertilizer (Triple Pro 15-15-15).

Microalgae are a rich source of proteins, pigments, and omega fatty acids and thus find application in human and animal feed production. *A. platensis* is one of the dominant species of microalgae used in the health food industry [69].The omega fatty acids from this microalga are used as human food and animal feed supplements. Phang et al. [70] found that the biomass composition of *Arthrospira* cultured in a high-rate algal pond for the treatment of sago starch processing wastewater can be used as high-quality animal feed, especially in the aquaculture industry. During the mentioned treatment of sago processing wastewater using *Spirulina*, COD, PO<sup>4</sup> 3−-P, and NH<sup>4</sup> + -N reductions of 94, 93, and 99%, respectively, were achieved [70].

Zainal et al. [71] reported that *A. platensis* was able to treat wastewater containing heavy metals and removed manganese by 84.9%; chromium by 83.8%; arsenic by 71.4%; nickel by 61.9%; zinc by 55%; copper by 52.8%, and iron by 45.1%.

Similar to bioconversion, microalgae could also carry out the biosorption of textile wastewater. For instance, *A. platensis* was used as a biosorbent to remove reactive red 120 (RR-120) from its aqueous solution. It achieved the maximum biosorption capacity of 482.2 mg/g removing 97% RR-120 from the solution [72].

#### *2.3.2. Oscillatoria tenuis*

+ )

grown in a BG 11 medium, an artificial medium normally used for *B. braunii* cultivation. And the algal hydrocarbon content was 23.8%, which was more than twice that in the BG 11 medium. Additionally, after the 26-day cultivation period, about 40.8% of TN and 93.3% of TP in ASLW were removed, also indicating good environmental benefits of algal bioremediation [65].

A study was conducted to evaluate the possibility of using wastewater from a soybean curd manufacturing plant as a growth promoter of *B. braunii* strain BOT-22. Soybean curd wastewater (SCW) was added to a AF-6 medium to set final concentrations at 0 (control), 1, 2, 5, and 10% (v/v). The growth and hydrocarbon production observed in the cultures with 1 and 2% SCW were significantly higher than that observed in the control. It was postulated that

The strain *Micractinium* sp. IC-76 was grown in municipal wastewater and showed a biomass productivity of 37.1 ± 4.1 mg/(L d) and a lipid content of 36.2 ± 0.1%, with a total content of saturated and monounsaturated fatty acids of 71.9%. The efficiency of nitrogen (N-NH<sup>4</sup>

*Micractinium* sp. IC-76 in the stationary phase of growth showed a significant difference in carbohydrate metabolism, especially sucrose concentration. High-lipid induction during cultivation in wastewater was also driven by changes in the biosynthesis of amino acids, fatty

*Micractinium* sp. Embrapa|LBA32 presented vigorous growth in a light-dependent manner in undiluted vinasse under non-axenic conditions. Microalgae strains presented higher biomass productivity in vinasse-based media when compared to standard BBM in cultures performed using 15 L airlift flat plate photobioreactors. Chemical composition analyses showed that proteins and carbohydrates comprise the major fractions of algal biomass. Glucose was the main monosaccharide detected, ranging from 46 to 76% of the total carbohydrate contents accord-

A recent study investigates the capacity of *I. galbana* in the bioremediation of aquaculture wastewater from a gray mullet *Mugil cephalus*. The experiment was conducted in batch conditions for 7 days using completely mixed bubble column photobioreactors. After 2 days, *I. galbana* removed 32 and 79% of dissolved inorganic nitrogen and dissolved inorganic phos-

It has been also reported that *I. galbana* cultured in open ponds has fatty acids and a high-

Phosphorus can be recycled from wastewater through microalgal cultivation and provided to crop plants in the form of microalgal biofertilizers. Guldhe et al. [21] reported filamentous

3−) removal was 96.4 ± 0.7 and 77.8 ± 5.6%, respectively. The strain

proteins and/or reducing sugars in SCW could enhance the growth [66].

*2.1.6. Micractinium genus*

68 Microalgal Biotechnology

and phosphorus (P-PO<sup>4</sup>

acids, and the tricarboxylic acid cycle [67].

ing to the culture media used [68].

phorus, respectively [10].

*2.3.1. Arthrospira platensis*

**2.3. Cyanobacteria**

**2.2. Haptophytes:** *Isochrysis galbana*

protein content, which are suitable for animal nutrition [20].

The performance of *O. tenuis* to remove nitrogen, phosphorus, and COD from secondary effluents of municipal domestic wastewater was investigated in batch experiments. *O. tenuis* had a biomass productivity of 150 mL/(L·d), a removal rate of NH<sup>4</sup> + -N of 96.1%, and total phosphorus and COD removal efficiencies of 82.9 and 92.6%, respectively, within 7 days at an aeration rate of 1.0 L/min [73].

At the same time, *O. tenuis* showed its capacity to remove reactive dyes from textile wastewater. This species degraded azo dyes into simple aromatic amines and decolorized dye wastewater [59].

#### **2.4. Binary and mixed culture systems**

Maintaining the uni-algal system requires a super clean environment, which can be attained under laboratory conditions only. In the outdoor cultivation of microalgae, it is almost impossible to maintain a uni-algal system. If so, it requires a lot of expertise and skills. Moreover, the biomass productivity of the uni-algal system is limited because of suppressed metabolic activity during night time or dark periods. Alternatively, heterotrophic microalgae are used, which are less sensitive to photoperiods, grow fast, and return high-biomass yields. However, a significant amount of CO<sup>2</sup> is produced during oxidative metabolism, which remains unused and is released into the environment. This CO<sup>2</sup> can be further utilized by employing autotrophic microalgae in the cultivation matrix. Therefore, the concept of a binary culture system arises [38]. Binary culture is considered superior to the uni-algal system in several different ways: binary culture can use wastewater as a nutrients source without sterilization unlike in single systems; microalgae observe a low level of contamination in binary culture because bacteria protect those invading pathogens; microalgae with increased growth rate would decrease the cultivation time and reduce the overall cost; binary culture also aids in bioflocculation and lipid induction; and so on [38].

microalgae, noting the low degradability of the cell wall, ammonium toxicity, and salinity as

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However, the use of microalgae as co-substrate is an approach to dilute complex compounds and balance the C/N ratio. Co-digestion has several advantages such as adjusting the C/N ratio, nutrients, and inhibitor compounds [34]. Ajeej et al. [77] also reported the increased activity of methanogenic microorganisms, a decreased anaerobic digestion inhibition by ammonium, and even increased cellulose activity when carbon-rich materials were added. Taking into account that the C/N ratio of the microalgal biomass is around 10:1 [78], the microalgae biomass can be considered as a suitable feedstock for carbon-rich substrates [79].

The main microalgae used for co-digestion have been described in the following paragraphs.

Ehimen et al. [80] added lipid-extracted *Chlorella* biomass resulting from microalga diesel production to glycerol (main by-product formed during the transesterification process) and observed an increase in the methane yield of 50% when compared to the digestion of residual

Wang et al. [81] used the biomass of microalga *Chlorella* sp. grown in laboratory culture for co-digestion with WAS. The batch experiments were carried out under mesophilic conditions with a working volume of 100 mL. Different volumes of algae and WAS were added to the digester. They experimentally proved that the addition of WAS improved the anaerobic digestion of the microalga *Chlorella*, producing 73–79% more methane than single microalga digestion. Similar results were obtained by Li et al. [82], who co-digested *Chlorella* sp. with chicken manure in batch experiments. The co-digestion enhanced the methane production obtained during the single digestion of chicken manure and *Chlorella* sp. by 14.20 and 76.86%, respectively. By contrast, Retfalvi et al. [83], using the same C/N ratio, but pretreating the

Beltran et al. [84] assessed the co-digestion of *C. sorokiniana* with WAS. Different co-digestion mixtures were tested in biochemical methane potential (BMP) tests under mesophilic condi-

25% microalga. This value was 22 and 39% higher than that obtained in the anaerobic digestion of the sole substrates, WAS and microalga, respectively. This mixture clearly improved

Rusten and Sahu [85] co-digested *Chlorella* sp. biomass and wastewater sludge (pretreated

compared to the anaerobic digestion of wastewater sludge alone. The co-digestion process achieved between 65 and 90% of specific methane gas production for sludge liquor depending on the HRT, temperature of incubation, and pretreatment of algae biomass. However, this study indicated that tested microalga could be cultivated in reject water to remove nitrogen

/g VS for the mixture 75% WAS and

/g VS fed) was not increased when

microalga, did not observe any positive effects on methane production.

anaerobic digestion by ensuring its viability, suitability, and efficiency.

tions. The highest methane yield obtained was 442 mL CH<sup>4</sup>

sludge liquor).The specific methane gas production (mL CH<sup>4</sup>

and phosphorus from the sludge liquor.

the main inhibitors of anaerobic digestion [76].

**3.1. Chlorophytes**

*3.1.1. Chlorella genus*

biomass alone.

Species selection is crucial for the success of microalgae cultivation in wastewater. Combining different species with varying metabolic potential would provide robustness to fluctuations in environmental factors and wastewater compositions, thereby giving more stability to the system. For instance, the potential application of microalgae consortia (*Chlorella* sp., *Scenedesmus* sp., and *C. zofingiensis*) compared to monoculture (*Chlorella* sp.) for the treatment of dairy wastewater was evaluated by Qin et al. [74]. They reported a significantly higher COD removal (57–62%) and phosphorous removal (91–96%) by microalgae consortia when compared to the monoculture of *Chlorella* sp. Furthermore, FAME profiles indicated that lipids produced from the microalgae consortia cultivation system were more suitable for biodiesel production [74].

In a very recent study [8], a mixed microalgae consortium (highly dominated by *Chlorella* species and small portions of *Scenedesmus* sp.) was cultivated using digestate (D), animal manure (AM), and textile wastewater (TW) as growth medium providing mainly N (nitrogen) and P (phosphorous) sources without any extra nutrient addition. After a cultivation period of 13 days, P was completely removed (100%); however, N was still remaining, and the removal rates of 70.1, 72.3, and 16.7% for TW, AM, and D, respectively, were achieved. The peak growth rate and biomass production of 0.419 d−1 and 0.4 g/L (in terms of volatile solids, VSs) were achieved using TW as growth medium [8].
