Keywords

1 Introduction

Astaxanthin, known as “super antioxidant,” can be obtained from synthetic and natural sources. Natural astaxanthin can be found in fishes (salmon), crustaceans (shrimp), Phaffia yeast and Paracoccus bacteria, zooplankton (krill), and some microalgae (e.g., Haematococcus pluvialis) (Higuera-Ciapara et al. 2006; Ranga Rao et al. 2014). H. pluvialis is produced commercially as the richest source of natural astaxanthin which has 20 times stronger antioxidant capacity than the synthetic astaxanthin (Lorenz 1999; Ranga Rao et al. 2010). Astaxanthin can be extensively applied in human nutrition, animal and aquaculture feed, and cosmetics industry.

Astaxanthin has high market value ($2500–7000/kg), and its market potentiality is estimated to increase from 280 metric tons, $447 million (in 2014), to 670 metric tons, $1.1 billion, by 2020 (Koller et al. 2014; Pérez-López et al. 2014; Industry Experts 2015). Presently, only <1% of the commercialized quantity is produced from H. pluvialis (Koller et al. 2014), and the interest of producing astaxanthin from H. pluvialis is increasing. Different approaches of production system have been reported such as photoautotrophic, heterotrophic, mixotrophic, indoor, outdoor, open raceway, photobioreactors, batch, fed-batch, two-stage mixotrophic, and attached biofilm-based system (Kang et al. 2005, 2010; Kaewpintong et al. 2007; Ranjbar et al. 2008; García-Malea et al. 2009; Issarapayup et al. 2009; Li et al. 2011; Han et al. 2013; Wang et al. 2013a, b; Park et al. 2014; Zhang et al. 2014).

The astaxanthin accumulation is controlled by various physicochemical factors such as temperature (Yoo et al. 2012), pH (Hata et al. 2001), light (Saha et al. 2013; Park et al. 2014), salinity (Kobayashi et al. 1993), plant hormones (Yu et al. 2015), and nutrient stress (Boussiba et al. 1999; Chekanov et al. 2014). Since wastewater contains (in)organic compounds, it can be a potential asset for different living creatures (Rogers et al. 2014). Microalgae have the ability to metabolize and eradicate pollutants, and also they predominate in breaking and separating resistant organic molecules from wastewater (Matamoros et al. 2016 a, b). Various bioproducts have been produced from microalgal biomass harvested during wastewater treatment (Woertz et al. 2014). Different investigations have verified the viability of utilization of microalgae in the treatment of wastewaters (municipal, agricultural, and industrial) (Chinnasamy et al. 2012; Fenton and hUallachain 2012; Dickinson et al. 2013; Neveux et al. 2016). Among them, municipal wastewater has the best potentiality for microalgae cultivation. Commonly, various culture media (BM, BG11, and M1B5) are used for cultivation of H. pluvialis, and for transformation of vegetative cells into cyst cells, different chemical additives such as ferric or acetate anions are used (Kobayashi et al. 1997; Ruen-ngam et al. 2010; Solovchenko 2013). Numerous experiments have been reported on the development of best synthetic medium (e.g., Gong and Feng 1997; Fábregas et al. 2000), but, as far as we are aware, a limited number of studies are accessible on the likelihood to use wastewaters for H. pluvialis cultivation and astaxanthin accumulation (Kang et al. 2006; Wu et al. 2013; Wang 2014; Sato et al. 2015; Ledda et al. 2015; Haque et al. 2016a; Liu 2018).

Recently, phyco-valorization (nutrient removal from wastewater and simultaneous by-product generation by microalgae) has gained great attention (Querques et al. 2015). H. pluvialis was explored for cultivation in diluted primary-treated sewage and primary-treated piggery wastewater which demonstrated better growth and successful uptake of nitrate and phosphorus (Kang et al. 2006). There are a lot of advantages of using wastewater such as reduction of costs and natural resource inputs and simultaneously obtainment of high-value bioproducts (Farooq et al. 2013), but there are a number of challenges involved too. The main challenges include the following:

  • Harvesting of the algae.

  • The control of biomass composition is complicated by the selection of the desired species.

  • Bacterial contamination.

  • Micro-pollutant removal.

  • The conceivable requirement for external CO2.

In this chapter, H. pluvialis-derived astaxanthin, its application and market potential, and culture conditions and nutritional requirements of H. pluvialis cell growth and astaxanthin formation have been discussed. The potentiality of microalgae cultivation using various wastewater streams and integration of H. pluvialis culture in different wastewater streams and nutrient removal and biomass production efficiency are also discussed. Furthermore, the challenges associated with coupling H. pluvialis cultivation in wastewaters and possible ways to overcome such challenges have been highlighted.

2 The Green Microalga H. pluvialis-Derived Astaxanthin

In H. pluvialis, maximum accumulation of astaxanthin can reach up to 5% DW (Wayama et al. 2013). Health food supplements consisting astaxanthin from microalgae considered as safe and broadly utilized as a nutraceutical supplement (Capelli and Cysewski 2013; Yang et al. 2013). H. pluvialis-derived astaxanthin can be used for health benefit in dosages from 3.8 to 7.6 mg per day (Yang et al. 2013). Due to structure, function, application, and security, H. pluvialis astaxanthin appears to be more effective than the synthetic one (Capelli and Cysewski 2013; Pérez-López et al. 2014; Shah et al. 2016).

2.1 Applications of Astaxanthin

Astaxanthin in Medical and Nutraceutical

Many published reports are available on human health and nutraceutical applications of astaxanthin (Guerin et al. 2003; Chew et al. 2004; Higuera Ciapara et al. 2006; Palozza et al. 2009; Yuan et al. 2011). It works as an antioxidant (Hussein et al. 2006; Liu and Osawa 2007; Ranga Rao et al. 2010), protects peroxidation of membrane lipids (Naguib 2000), terminates the induction of inflammation, helps in ulcer disease (Liu and Lee 2003), enhances human digestive health (Nishikawa et al. 2005; Kamath et al. 2008), and deals with treatment of gastrointestinal pain (Andersen et al. 2007; Kupcinskas et al. 2008).

Astaxanthin can be helpful for reduction of risk for heart attacks (Iwamoto et al. 2000), increment of basal arterial blood flow (Miyawaki et al. 2008), and reduction of blood plasma level (Karppi et al. 2007). It can also reduce the effects of Alzheimer’s and neurological disorders; hinder fibrosarcoma growth, cancer cells (breast and prostate), and embryonic fibroblasts (Palozza et al. 2009); and improve respiratory and sympathetic nervous system (Nagata et al. 2006) and mammary tumor (Nakao et al. 2010).

Astaxanthin also helps to protect the skin from photooxidation by UV induction and has antiaging effects (Seki et al. 2001; Yamashita 2002; Tominaga et al. 2012; Ranga Rao et al. 2013). In the case of human, astaxanthin can improve semen quality, pregnancy rate, and sperm velocity (Elgarem et al. 2002; Comhaire et al. 2005) and decrease unexplained infertility (Andrisani et al. 2015).

Astaxanthin in Aquatic Animal and Poultry Diet

Haematococcus-derived astaxanthin can provide essential nutrient for body weight increment and breeding of economically important fishes such as salmonid, red sea bream, rainbow trouts, and shellfish (shrimp). It has been proved as important compound for improvement of pigment in the fish flesh (Torrissen and Naevdal 1984; Tolasa et al. 2005). Use of H. pluvialis biomass has shown to enhance egg quality, growth, and rate of survival of fish (salmonid, sea bream, and rainbow trout, ornamental fish), fry (Arai et al. 1987; Ako and Tamaru 1999; Sommer et al. 1991; Choubert and Heinrich 1993; Sheikhzadeh et al. 2012a, b), and shrimp (Arai et al. 1987; Parisenti et al. 2011). It has been demonstrated that the diet containing H. pluvialis improved the growth of adult yellow croaker fish (Li et al. 2014). H. pluvialis appeared to be effective in egg yolk coloration, improving egg-laying capacity in hen (Elwinger et al. 1997), muscle in meat-producing chicken (Inborr and Lignell 1997; Inbbor 1998), and fertility and decreasing mortality of chicken (Lignell and Inborr 1999, 2000).

2.2 Market Potential of Astaxanthin

Recently, there has been increasing pattern toward utilizing organic in food, feed, and cosmetic products. The interest for H. pluvialis astaxanthin in the international market worldwide has been “emerging” as a result of expanding customer attention to its medical advantages. Worldwide market for astaxanthin (synthetic and natural) is assessed in 2014 at 280 metric tons which is anticipated to achieve by 2020 at 670 metric tons (Industry Experts 2015; Panis 2015). The market value of astaxanthin is about $2500–7000/kg, and in some cases for H. pluvialis astaxanthin, it goes up to $15,000/kg (Borowitzka 2013; Koller et al. 2014; Pérez-López et al. 2014; Industry Experts 2015). Natural astaxanthin is 3–4 times more expensive than the synthetic one (Han et al. 2013). Considering the increasing market potentiality for natural astaxanthin for industrial utilization, large-scale production of H. pluvialis has great prospects and appealing commercial possibility. However, contemporary market requirement for astaxanthin from H. pluvialis is not fulfilled. Once the production technology is optimized, the production costs H. pluvialis astaxanthin would be comparable to the artificial astaxanthin (Pérez-Lópezetal et al. 2014).

3 Culture Parameters for H. pluvialis Growth and Astaxanthin Production

Improvement of culture conditions is important to accomplish greater yield and astaxanthin generation. These conditions have diverse optimum level for cell growth and pigment production. Different kinds of media such as BG-11, BBM, OHM, and KM1-basal medium (Bischoff and Bold 1963; Rippka et al. 1979; Kobayashi et al. 1993; Fábregas et al. 2000) are used for cultivation. At nutrient-deficient conditions, astaxanthin accumulates inside the cells (Saha et al. 2013). In nitrogen-deficient condition, the production rate of astaxanthin is twice than the limitation of phosphorus. Micronutrients (selenium and chromium) play important role to increase yield and astaxanthin formation (Tripathi et al. 1999; Fábregas et al. 2000; Domínguez-Bocanegra et al. 2004). Astaxanthin generation can also be accelerated by incorporating 0.25–0.5% w/v of NaCl or combining 2.2 mM sodium acetate to the media (Sarada et al. 2002b).

The appropriate temperature for H. pluvialis ranges from 20 to 28 °C (Fan et al. 1994; Hata et al. 2001; Lababpour et al. 2005; Kang et al. 2010; Yoo et al. 2012; Wan et al. 2014a). However, >30 °C temperature triggers a transition from green to red stage (Tjahjono et al. 1994). pH also can significantly have an effect on the growth and synthesis of carotenoids. The optimum pH ranges from 7.00 to 7.85 (Hata et al. 2001; Sarada et al. 2002a). The optimal light irradiation ranges from 40 to 50 μmol photons m−2 s−1 (Hata et al. 2001; Chekanov et al. 2014; Park et al. 2014). Optimum light intensity to accomplish better growth rates inclines to be greater such as 70 (Zhang et al. 2014), 80 (Saha et al. 2013), 90 (Fan et al. 1994), or 177 μmol photons m−2 s−1 (Domínguez-Bocanegra et al. 2004). During green stage, the regular photoperiod (12:12 or 16: 8 h) is frequently maintained (Saha et al. 2013; Park et al. 2014) but higher growth obtained with continuous light (Domínguez-Bocanegra et al. 2004).

Culture Systems

H. pluvialis can be grown indoor and outdoor and in open or closed system; batch, fed-batch, semicontinuous, or continuous system; and photoautotrophic, heterotrophic, or mixotrophic modes.

Photoautotrophic Culture

This type of culture is generally performed in ponds/raceways or photobioreactors. Typically tubular, bubble column and airlift photobioreactors are used for cultivation. Since circumstances for maximum cell yield and astaxanthin concentration are usually incompatible, a double-step production policy is frequently followed for the industrial cultivation. The step one is to maximize vegetative growth in optimum conditions (e.g., less light intensity and with nitrogen) (Boussiba 2000; Aflalo et al. 2007; Del Rio et al. 2007). Once maximum growth is achieved, in the second step, the cells moved to stress situation (e.g., strong light and nitrogen limited, pH or salt manipulation, phosphate depletion, etc.). These stress conditions either individually or in combination with others can stimulate astaxanthin formation (Fábregas et al. 2001; Torzillo et al. 2003; Orosa et al. 2005; He et al. 2007; Hu et al. 2008; Li et al. 2010; Choi et al. 2011). The biomass production in vegetative and red stage varied from 0.01 to 0.5 g L−1 d−1 and 0.01 to 4.8 g L−1 d−1, respectively. In terms of astaxanthin production and content, it varied from 0.44 to 21 mg L−1 d−1 and 0.8 to 4.8% of DW, respectively (Table 1). Attached cultivation strategy is utilized in the initiation of astaxanthin formation in H. pluvialis. In this system, the biomass and astaxanthin productivities were 2.8- and 2.4-fold greater than those of the suspended cultivation system, respectively (Wan et al. 2014b). Additional researches that used the same techniques have shown increased astaxanthin production: 124 mg m−2 d−1 (Yin et al. 2015) and 164.5 mg m−2 d−1 (Zhang et al. 2014). Attached induction system can be a potential way to enhance commercial profit and significantly lower cultivation cost (Zhang et al. 2014; Wan et al. 2014b). Park et al. (2014) invented “perfusion culture” system coupling it with stepwise increase associated with light intensity. This culture can offer greater cell growth of 0.18 g L−1 d−1. Under stepwise improved light irradiance (150–450 μE/m2/s), cell growth of 12.3 g L−1 can be achieved. This cell growth is usually 3.09 and 1.67 times greater than batch and fed-batch processes, respectively (Park et al. 2014).

Table 1 Biomass production and astaxanthin content of H. pluvialis grown in various studies
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Heterotrophic and Mixotrophic Culture

In heterotrophic growth, organic compounds act as carbon and energy for cell propagation and secondary metabolite construction with the absence of light. Different sources of organic carbon have been utilized in this culture. It was shown that acetate helped effectively to cyst formation and astaxanthin formation (Kobayashi et al. 1991; Kakizono et al. 1992; Orosa et al. 2000; Hata et al. 2001; Kang et al. 2005). These microalgae can be also cultured mixotrophically utilizing acetate or carbohydrates (Kobayashi et al. 1993). It is proved that biomass and astaxanthin accumulation can be improved following this culture system. For example, cell density of 0.9–2.65 g L−1 and astaxanthin content of 1–2% DW were achieved (Chen et al. 1997; Wang et al. 2003). A sequential, heterotrophic-photoautotrophic culture strategy was also investigated. Biomass was produced by utilizing heterotrophic culture, but for astaxanthin production, photoautotrophic culture was applied under nitrogen depletion, with bicarbonate or CO2 as carbon sources. In this system, a superior astaxanthin content (7% DW) was obtained which is 3.4-fold higher than heterotrophic induction (Kang et al. 2005).

4 Wastewater as a Resource for Microalgae Cultivation

Microalgae cultivation and biomass production require huge quantities (for 1 gram dry biomass >1 kg water) of water (Burlew 1953; Shen 2014). Wastewater (cheap and readily available) provides appropriate atmosphere (pH, dissolved CO2, and HCO3 −) and macronutrients (nitrate, ammonia, phosphate) and micronutrients that support for microalgal growth (Abdel-Raouf et al. 2012; Ji et al. 2013; Ajayan et al. 2015; Ding et al. 2015). Wastewater-grown microalgae biomass can be used to extract the accumulated nutrients (Mehta et al. 2015; Gouveia et al. 2016). Three nutrients (carbon, nitrogen, and phosphorus) are of most interest during evaluating a wastewater for microalgae growth enhancement (Kabra et al. 2014).

4.1 Macroelements and Microelements

The cell growth and biochemistry of microalgae require the receptiveness of 15–20 essential elements. The macronutrients consist of C, N, P, H, O, S Mg, K, Na, and Ca, and the micronutrients include Fe, Cu, Mn, Zn, Cl, V, Mo, B, Co, and Si (Eyster 1964). The macroelements are typically utilized as development materials, and the microelements are involved in biological reactions (Arnon 1961). Five microelements (Mn, Zn, Cu, Ca, and Fe) are directly associated with microalgal photosynthesis. Microelements (Cl and Mn) play an important role in O2 evolvement. The supplementation of macroelements (C, N, and P) with essential microelements (Si, Mg, Ca, Fe, P, S, Mn, Zn, Cu, and Co) is needed for continuous microalgae growth. In case of application of wastewater for microalgae cultivation, the supply of essential microelements such as Si, Mg, Ca, Fe, P, S, Mn, Zn, Cu, and Co rarely limits microalgal growth.

4.2 Composition of Wastewater

Wastewater is a compound mixture of organic, inorganic, and artificial elements. Three quarters of organic carbon in sewage are proteins, carbohydrates, fats, amino acids, and volatile acids. The inorganic parts include large amount of calcium, potassium, sodium, magnesium, chlorine, sulfur, phosphate, bicarbonate, ammonium, and heavy metals (Lim et al. 2010). Wastewaters from various sources (municipal, agricultural, and industrial) can be treated efficiently by microalgae. The typical N:P features of different wastewaters and the feasibility of cultivation of microalgae are shown in Table 2.

Table 2 Comparison between the physicochemical characteristics of wastewaters and a common synthetic medium
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In the wastewater influent, nitrogen is present in the form of ammonia (NH4+), nitrite (NO2−), or nitrate (NO3−). Phosphorus is present as phosphates (PO43−) in wastewater. Municipal wastewater contains several heavy metal pollutants such as arsenic, cadmium, chromium, copper, lead, mercury, and zinc (European Commission on Environment 2002). It contains comparatively lower amounts of total N and P (10–100 mg L−1) (Dela Noue et al. 1992). Once the secondary treatment is done, total N and P decrease to 20–40 mg L−1 and 1–10 mg L−1, respectively (McGinn et al. 2011), which is very suitable for microalgae growth. The N and P ration in municipal wastewater is about 11 to 13 (Christenson and Sims 2011). The widely accepted N:P ratio for microalgae growth is 16 (Larsdotter 2006; Christenson and Sims 2011; Park et al. 2011; Cai et al. 2013), on the basis of empirical formula C106H181O45N16P (Stumm and Morgan 1970). The typical microalgae cell biomass contains 6.6% N and 1.3% P in dry weight (Chisti 2013) with a molar N:P ratio of 11.2, which is similar to that found in wastewater.

Agricultural wastewater derived from animal manure contains N and P concentrations of >1000 mg L−1 (Dela Noue et al. 1992). Agricultural runoff consists of herbicides, fungicides, and insecticides.

Industrial wastewater contains less N and P compared to agricultural and municipal wastewater. It has high levels of heavy metal pollutants such as Cr, Zn, and Cd and organic chemical toxins such as hydrocarbons, biocides, and surfactants (Chinnasamy et al. 2010). Textile, tanning, leather, and electroplating and related metal processing industry effluent possess considerable amounts of toxic metal ions (Salama et al. 2017).

4.3 Treatment of Wastewaters

Municipal Wastewater Treatment

Increasing urbanization and population expansion have resulted in large quantities of municipal wastewaters produced every day. Physical and chemical treatment methods are commonly used for removing buoyant, non-buoyant, and dissolved organic materials from wastewaters (Ruiz-Marin et al. 2010). Microalgae cultivation into the municipal wastewater treatment systems for nutrient removal has been widely studied. For example, Pittman et al. (2011) reported that Chlorella sp. and Scenedesmus sp. performed with very high efficiency on nutrient removal in sewage wastewater after secondary treated, particularly ammonia, nitrate, and total P, ranging from 80% to 100% removal rates in many cases. Another study indicated that C. vulgaris could remove more than 90% of N and 80% of P from primary-treated municipal wastewater (Lau et al. (1995). Dunaliella salina showed the capacity for removing nitrate, ammonia, and phosphorous in the range of 45–88% from municipal wastewater after a 6-day cultivation (Liu and Yildiz 2018).

Agricultural Wastewater Treatment

Agriculture is another major wastewater-producing sector. Agricultural wastewater is frequently derived from livestock production and contains high levels of N and P (Wilkie and Mulbry 2002). Generally, livestock manure is often treated and used as fertilizer. However, nutrients may not be completely consumed due to the various ratios of N:P requiring by the crops. As a result, excess nutrients find their ways to the surrounding aquatic systems and cause eutrophication significantly reducing water quality (Cai et al. 2013). Piggery wastewater is typically treated with anaerobic bacteria for reduction of nutrient. However, the nutrient removal capacity of anaerobic bacteria is comparatively lower than microalgae and some cyanobacteria (Markou and Georgakakis 2011). As in the case of municipal wastewaters, previous researches have also demonstrated that microalgae can significantly assimilate N and P from manure-based wastewaters. For example, An et al. (2003) reported that 80% of nitrate content was effectively removed from piggery wastewater by Botryococcus braunii. Moreover, compared with microalgae that were cultivated in municipal wastewaters, Wilkie and Mulbry (2002) indicated that higher microalgae growth rates and equivalent nutrient removal efficiencies were observed in manure-added recycling wastewater.

Industrial Wastewater Treatment

The traditional methods of industrial wastewater treatments include electrowinning, precipitation, and ion exchange. Since industrial wastewaters contain lower N and P contents and greater levels of toxic elements, most microalgae cannot grow well. It is necessary to select specific strains that have high metal absorption capacities to handle industrial wastewater remediation. So far, only a few strains have been explored for metal removal capacity research. One study using carpet mill wastewater, which has relatively lower toxins and higher N and P contents, reported that Botryococcus braunii, Chlorella saccharophila, and Pleurochrysis carterae grew well in untreated wastewater with large amounts of biomass generated (Chinnasamy et al. 2010).

5 Integrating H. pluvialis Cultivation in Wastewater Treatment and Nutrient Removal

The growth rate of H. pluvialis is slow, and its cultivation is a highly sensitive process due to its susceptibility to contamination by other algae and microbes (Orasa et al. 2000). Generally, BM, BG11, and M1B5 media used for cultivation of H. pluvialis and chemical additives such as ferric or acetate anions are added to stress the cells (Kobayashi et al. 1997; Ruen-ngam et al. 2010; Solovchenko 2013).

Although various researches have been performed on the development of optimal synthetic growth medium (e.g., Gong and Feng 1997; Fábregas et al. 2000), only few studies focused on the possibility to use wastewaters for H. pluvialis and succeeding astaxanthin production (Table 3). For instance, Kang et al. (2006) reported H. pluvialis cultivation in primary-treated wastewater and piggery wastewater. They showed that the cell growth rate in primary-treated wastewater was 0.24 day−1, which was comparable to 0.23 day−1 in artificial medium; the cells were composed of 5.1 and 5.9% astaxanthin content using the two-step process; and the cells yielded 43 mg L−1 nitrogen and 2.6 mg L−1 phosphorus.

Table 3 Research highlights on integration of different wastewaters with H. pluvialis
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Compared with most microalgal species reported in the literature, H. pluvialis attained highest biomass production (27.8 mg L−1 d−1), efficient nutrient removal (both nitrogen (93.8%) and phosphorus (97.8%) were removed efficiently), and highest lipid accumulation (43%) in unsterilized domestic secondary effluent (Wu et al. 2013).

Sato et al. (2015) reported a new wastewater treatment process that involves coagulation, ozonation, and microalgae H. pluvialis cultivation. H. pluvialis grew well in the supernatant of coagulated wastewater, and the astaxanthin yield was 3.26 mg/L, and total phosphorus and nitrogen contents decreased to 99% and 90%, respectively.

In another study, wastewater treatment and astaxanthin production were conducted by a primary treatment filtering system: culture and subsequent carotenogenesis induction of H. pluvialis on piggery wastewater.

In this study, a drastic reduction in macro- and micronutrient concentration (up to 99% for NO3-N and NH4-N, 98% for TP) and astaxanthin accumulation of 1.27% on a dry weight were observed (Ledda et al. 2015). This method showed potentiality as biological wastewater treatment process since it can combine inorganic waste removal without any additives and the simultaneous production of astaxanthin.

Since H. pluvialis can use CO2, CO3, and carbohydrates as carbon sources, its production cost can be reduced by utilizing waste sources like flue gasses or waste containing carbon and nutrient compounds (Wu et al. 2013).

The required energy and nutrients in auto-, hetero-, and mixotrophic cultivation can be recycled from anaerobic digestion. Based on the culture system, carbon sources can vary. The recycled CO2 from energy production at anaerobic digestion can be used in photoautotrophic cultivation. The required carbon (carbohydrates or acetate) can be provided from alternative source in heterotrophic cultivation. These carbon sources can be produced from waste (carbohydrate-rich food waste from food industry can be used in heterotrophic cultivation) (Wang 2014). In mixotrophic cultivation both carbon sources can be utilized. After concurrent extraction of astaxanthin and triglycerides, algal cake is used as a feedstock for biogas production through anaerobic digestion that helps in the extraction of residual energy from this integrated bioprocess (Shah et al. 2016). The biorefinery strategy is shown on Fig. 1.

Fig. 1
figure 1

Schematic diagram of two-stage cultivation H. pluvialis biorefinery producing astaxanthin and either edible oil or biofuel compound-biodiesel. (Adapted from Shah et al. 2016). Green stage: performed using either photoautotrophic (deep green section) or hetero−/mixotrophic (pale green section) systems. Red stage: (red section) takes place after green stage to maximize astaxanthin content. Recycling of waste is performed through an aerobic digestion process. The following annotations are used: solid arrows, subsequent steps; dashed arrows, optional steps; double lines, final products; double arrows, inputs; dotted lines, opportunities for recycling resources

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In a recent study by Haque et al. (2017), high-density (4.37 g/L) H. pluvialis culture was obtained using the bioethanol plant waste stream as the growth media and resulted in 91.67% total nitrogen and 100% total phosphorous removal. The residual microalgal biomass, obtained after astaxanthin extraction (1.109 mg/g DW), was characterized as a potential bioenergy feedstock. This production process could be environmentally friendly and economically viable, compared to conventional astaxanthin production processes, due to integrating culture in an existing bioethanol plant and using the waste product produces in the plant. Culturing H. pluvialis in bioethanol wastewater streams can be a greener alternative to conventional media. The maximum vegetative growth of H. pluvialis was obtained in 60× diluted thin stillage, and maximum astaxanthin production was obtained in GroAst media (60% 60× thin stillage and 40% acetate-rich process condensate). The GroAst media appeared to be not only a cheaper media, compared to the chemically synthesized media, but it is also a “greener” sustainable alternative to conventional growth media (Haque et al. 2016b).

Minkery wastewater contains extremely high level of ammonia, which is a different N source from BBM. H. pluvialis grew well in the appropriately diluted minkery wastewater (MW) media, and a higher biomass production was realized as compared with conventional culture medium under optimal growth condition. H. pluvialis achieved maximum biomass at 1.5% MW cultures, yielding 906.03 ± 34.0 mg L−1, with a successful removal of total nitrogen and phosphorus in a 6-day culture. The optimal initial cell density and volume ratio between microalgae and MW were also determined to have great help on maximizing the biomass yield. The findings support the claim that integration of wastewater into microalgae cultivation has the advantages of reducing cultivation costs and natural resource inputs and simultaneously obtaining high-value bioproducts (Liu 2018).

Considering the above facts, H. pluvialis can be considered as a promising candidate for integrated systems of wastewater treatment and microalgae cultivation while producing high-value bioproducts.

6 Challenges Associated with Growing H. pluvialis in Wastewater Streams

Despite the promising features of microalgae, there are huge challenges to overcome before this route can be exploited in commercially and environmentally sustainable manner. The following points are considered as the most important challenges for the cultivation of microalgae in general and H. pluvialis using wastewater:

  • The cultivation of H. pluvialis in wastewater can be susceptible to contamination by fungus, zooplankton (rotifer), protozoans (e.g., amoebas, ciliates), and other microalgae due to its relatively slow growth (Han et al. 2013; Orasa et al. 2000).

  • Abiotic contaminants in wastewater such as CO2, NOx, SOx, O2, and NH3+ and heavy metals can also inhibit microalgae like H. pluvialis growth (Kumar et al. 2010).

  • In case of low concentration of trace mineral nutrients in the wastewater, it can result in poor growth, low biomass, and low lipid productivity (Christenson and Sims 2011). However, Kang et al. (2007) and Hata et al. (2001) indicated that high concentration of nutrients would also cause inhibitory effects on H. pluvialis growth, and thus, the suitable concentration of wastewater must be determined for H. pluvialis cultivation.

  • Due to the lack of carbon sources in most domestic wastewater, the growth of the microalgae can be inhibited which might eventually affect the treatment of the wastewater (Craggs et al. 2011).

  • High concentration of oxygen in wastewater can induce oxidative damage to microalgae cell and inhibit photosynthesis (Christenson and Sims 2011).

  • The cost and energy demand of harvesting microalgae in general and H. pluvialis from wastewater either by flocculation or centrifugation are still very high (Razon and Tan 2011; Acién et al. 2012; Shah et al. 2016).

7 Conclusion and Prospects

This chapter provides perception regarding the recent scientific and technical improvement in different areas of H. pluvialis-derived astaxanthin, its application and market potential, and culture conditions and nutritional requirements of this microalgal cell growth and astaxanthin formation. It also scans a broader image including the potentiality of microalgae cultivation using various wastewater and integration of H. pluvialis culture in different wastewater streams and nutrient removal and biomass production efficiency to the challenges associated with growing H. pluvialis in wastewater streams.

Recently the demand from H. pluvialis is increasing. A number of developments have been obtained concerning production and processing to achieve astaxanthin during the last decade. Still its large-scale cultivation is very expensive for mass adoption of natural astaxanthin compared to the synthetic one. H. pluvialis has been cultured in different ways. Various studies have been focused on optimization of parameters (media, light, pH, temperature, etc.) for maximum growth. For biomass accumulation and astaxanthin production, most of these parameters found different.

There is not much can be done to solve this challenges since it is fundamentally connected with the whole life cycle of H. pluvialis. We believe that integration of H. pluvialis cultivation with wastewater treatment could be a great option to produce astaxanthin effectively in large scale. Research in the use of wastewater for cultivating H. pluvialis is still very limited as compared to research for growing other microalgae species. Therefore, further clarifications are needed to prove the feasibility of H. pluvialis-based systems in full scale. A number of wastewater types are encouraged to be investigated in Haematococcus cultures. Moreover, the relevant optimal production routes and advances in technologies are needed. The improvements in integration processes, harvesting, and extraction technology will contribute to accelerate the speed of the Haematococcus-derived astaxanthin production from laboratory scale to commercial scale. Further study in these areas can have a profound influence on the market of natural astaxanthin from H. pluvialis.

The challenges of wastewaters directly for H. pluvialis culture should be addressed since they restrict the utilization of the easily accessible and low-cost wastewater. There are a number of areas that can improve the integrated H. pluvialis cultivation using wastewater for nutrient removal and efficient astaxanthin production. These include the following:

  • Consideration of sterilized wastewater for microalgal cultivation to prevent biotic contamination.

  • Coupling of immobilize or attached cultivation of H. pluvialis in wastewater stream to maximize the biomass production.

  • In cases of low concentration of nutrients, there is a need to supplement these nutrients in wastewater to achieve high productivity.

  • Bubbling of CO2 can improve algae growth while using domestic wastewater with lack of carbon source for H. pluvialis cultivation.

  • Technological advancement in cost-effective H. pluvialis biomass harvesting from large-scale wastewater culture to make it more economically attractive.

Future improvements in these fields can have a thoughtful effect on the commercial implementation of H. pluvialis astaxanthin products. Finally, global microalgae industry can be benefited in the near future.