Keywords

1 Introduction

Chlorella is a group of eukaryotic green microalgae with high photosynthesis ability. Through efficient photosynthesis, Chlorella is able to reproduce itself within several hours, requiring only sunlight, carbon dioxide, water, and a small amount of nutrients. Chlorella is easy to grow, has a simple life cycle and metabolic pathways similar to higher plants, and thus has long been employed as a model organism to investigate the mechanisms of photosynthesis and carbon dioxide assimilation (Calvin–Benson cycle). Due to its high protein content and richness in carotenoids, vitamins, and minerals, Chlorella has also long been proposed as a food substitute for humans and is now widely produced as health food in Germany, China, Japan, and several other Asian countries. Recently, because of the energy crisis and public interest in green renewable fuels, Chlorella has been cited as a promising candidate feedstock for biofuel production and has gained increasing scientific and industrial attention in that it grows fast, has high oil content, and outdoor mass cultures are easy to maintain. This chapter gives an overview of Chlorella, the industrially important microalgal genus, covering its taxonomy, growth physiology, cellular chemical composition, mass cultivation, and potential products and applications. An integrated production of biofuels and other bioproducts coupled with the treatment of greenhouse gas and wastewater is proposed, which may offset the Chlorella-based production cost while providing significant environmental benefits.

2 Morphology, Ultrastructure, and Taxonomy

Chlorella is a genus of unicellular and nonmobile green algae. Chlorella vulgaris is the type species of this genus, which was first described by M.W. Beijerinck in 1890. Commonly, Chlorella cells are spherical or ellipsoidal and the cell size may range from 2 to 15 μm in diameter. They are widely distributed in diverse habits such as freshwater, marine water, soil, and are even symbiotic with lichens and protozoa. Chlorella has no sexual life cycle and reproduces itself through asexual autospore production. When mature, autospores are simultaneously released via rupture of the mother cell wall. The number of autospores derived from a single mother cell may vary greatly from 2 to 16.

The ultrastructure of Chlorella has been studied extensively in past years. Under transmission electron microscopy, the visible structures within the cell include the chloroplast, nucleus, mitochondria, vacuole, starch, lipid bodies, and so on [1–3]. Generally, Chlorella has a cup-shaped chloroplast located peripherally in the cytoplasm. The nucleus situates near the cytoplasmic membrane, and the mitochondria are closely associated with the chloroplast. Pyrenoid, a conspicuous and easily recognizable structure, is present in most of the Chlorella species [2–4]. Usually, the pyrenoid is centrally located in the chloroplast and surrounded by the starch sheath [3]. In some Chlorella species, the pyrenoid contains many lipid-containing globules that are known as pyrenoglobuli and may function as secondary storage products [3, 4]. Chlorella has a thick and rigid cell wall, but the cell-wall structure may differ greatly across the species [2, 5]. When transferred to stress conditions (e.g., nitrogen starvation), the cell wall thickens and the chloroplast begins to regress to the proplastid stage with a gradual reduction in thylakoid number, accompanied by the accumulation of lipid bodies in the cytoplasm [3].

To date, there are hundreds of Chlorella strains reported in the literature, but the classification of Chlorella has been problematic due to the lack of conspicuous morphological characters. Kessler [6] proposed a sound taxonomic method for Chlorella based on multiple biochemical and physiological characters, that is, hydrogenase, secondary carotenoids, acid or salt tolerance, lactic acid fermentation, nitrate reduction, thiamine requirement, and the GC content of DNA. By comparing these characters, 77 Chlorella strains from the Collection of Algae at Göttingen (SAG, Germany) were assigned to 12 taxa and Chlorella was suggested to be an assembly of morphologically similar species of polyphyletic origin. Afterwards Kessler and Huss [7] examined 58 Chlorella strains from the Culture Collection of Algae at the University of Texas (UTEX, USA) according to the above-mentioned biochemical and physiological characters and reassigned them to 10 well-established species. The sugar composition of the cell wall (either glucosamine or glucose and mannose) has also been used for Chlorella classification [8, 9]. In addition, Huss et al. [10] examined the Chlorella genus by using a phylogenic approach based on complete 18S rRNA sequences, and considered it as a polyphyletic assemblage dispersed over two classes of Chlorophyta, that is, Chlorophyceae and Trebouxiophyceae; only four species were suggested to belong to this genus: Chlorella vulgaris, Chlorella sorokiniana, Chlorella kessleri, and Chlorella lobophora. Recently, based on the sequences of 18S rRNA and ITS2 region, Krienitz et al. [11] further investigated the phylogenesis of Chlorella and suggested the exclusion of Chlorella kessleri from the Chlorella genus. In this chapter, we regard Chlorella to be the Chlorella sensu lato and include the data of Chlorella species that may have been excluded from the genus by the above-mentioned studies.

3 Growth Physiology

Chlorella is able to convert solar light energy to chemical energy through efficient photosynthesis. Similar to C3 higher plants, photosynthesis in Chlorella consists of light-dependent reactions and carbon dioxide fixation. The light-dependent reactions produce high-energy molecules ATP and NADPH, which are utilized in the Calvin–Benson cycle to fix carbon dioxide. Chlorella performs photosynthesis efficiently at a relatively low light intensity and becomes saturated when the light intensity reaches a certain value, which may range from 80 to 400 µE m−2s−1on a per cell basis [12, 13]. Higher light intensity above the saturation value may inhibit the photosynthesis of the algae and even cause the destruction of chlorophylls (photobleaching) and cell deaths. The incident intensities of solar light can reach up to 2,500 µE m−2s−1, much higher than that required for photosynthesis saturation. Most of the sunlight energy is lost as heat and only a small portion can be converted to chemical energy by photosynthesis. Although the theoretical maximum solar energy conversion efficiency of oxygenic photosynthesis is thought to be around 8–10 % [14], the outdoor culture of Chlorella can only achieve a low photosynthetic efficiency (PE), for example, 2.7 % in full sunlight [15]. The yet to be enhanced PE remains a big challenge for outdoor mass cultures of algae and presents a promising direction toward the increase of biomass production.

Chlorella growth requires nutrients including carbon, nitrogen, phosphorus, sulfur, and metals. Carbon is the predominant element of Chlorella and carbon dioxide is the primary carbon source for photoautotrophic growth of Chlorella. Chlorella utilizes carbon dioxide principally in the undissociated form of CO2 or H2CO3 [16]. The atmospheric air contains only 0.04 % CO2, which is not sufficient to maintain rapid growth of Chlorella for high cell density. Therefore, a supply of air enriched with CO2 at the concentration of 1–5 % is usually provided to Chlorella cultures [17, 18]. Higher levels of CO2 may cause a decrease in pH of the medium and thus inhibit or even block the algal growth [19, 20]. Nevertheless, the high-CO2–tolerant Chlorella species as reported by Papazi et al. [21] and Sakai et al. [22] can grow well in the presence of up to 40 % CO2, although the optimal growth is obtained under lower CO2 concentrations. Nitrogen is the second most important element in Chlorella. Generally, Chlorella is able to utilize nitrate, ammonia, and organic sources of nitrogen such as urea, glycine, and amino acids [17, 23, 24]. Both nitrate-N and urea-N cannot be directly incorporated into organic compounds by Chlorella and first have to be reduced to ammonia-N. Ammonia and urea are economically more favorable than nitrate as nitrogen sources in that the latter is more expensive per unit N. The uptake of ammonia may result in acidification of the medium, nitrate may cause alkalinization, whereas urea leads to only minor pH changes [25]. In this context, urea is the better choice of nitrogen source, avoiding a large pH shift of unbuffered medium. Different Chlorella species may favor different nitrogen sources for growth, for example, Chlorella pyrenoidosa prefers urea to nitrate or glycine for boosting biomass production and Chlorella protothecoides gives higher biomass yield when using nitrate rather than urea as the nitrogen source [23, 26]. Nitrogen concentration in the culture medium plays an important role in regulating algal growth and metabolism. Nitrogen deficiency/starvation retards the growth of Chlorella, causes the decrease of protein levels, and promotes the accumulation of lipids within cells (Illman et al. [27]; Ördög et al. [28, 29]). Phosphorus is the third essential nutrient required for normal growth of Chlorella. It is involved in the formation of nucleic acid and cell membrane, as well as of ATP that provides energy for cellular metabolism. The most used phosphorus source for algal cultivation is phosphate, either as H2PO4 1− or HPO4 2−. Sulfur is an indispensable constituent of some essential amino acids, vitamins, and sulfolipids. Usually, it is provided in the form of sulfate for algal growth. Other inorganic nutrients include K, Ca, Mg, Fe, Cu, Zn, Mn, and Mo, among others, needed in trace amounts.

In addition to photoautotrophy, Chlorella is able to utilize organic carbon sources alone or together with CO2 and light for heterotrophic or mixotrophic growth (Table 1). Sugars are the most conventional organic carbon sources widely used for Chlorella fermentation [30–32]. Liu et al. [30] surveyed the utilization of various monosaccharides and disaccharides by Chlorella zofingiensis, indicating that glucose, fructose, mannose, and sucrose were able to be efficiently consumed by C. zofingiensis for fast growth whereas lactose and galactose were poorly assimilated. The growth of C. zofingiensis was inhibited when the sugar concentration exceeded 20 g L−1 as indicated by the decreased specific growth rate [33]. Raw materials rich in carbohydrates such as artichoke tubers and sorghum stems have also been reported as carbon sources to feed heterotrophic Chlorella cells [34, 35]. Other organic carbon sources that can be used for heterotrophic Chlorella growth include acetate, glycerol, lactate, glutamate, and methanol (Table 1). The high concentrations of these organic carbons, however, may confer an inhibitive effect on Chlorella growth; for example, glycerol above 2 % was reported to inhibit the growth of Chlorella vulgaris [36] severely, and methanol above 1 % was lethal to Chlorella minutissima [37].

Table 1 Selected Chlorella species reported for mixotrophic and/or heterotrophic growth
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4 Mass Cultivation

The outdoor mass cultivation of Chlorella started in the late 1940s with the almost concurrent launch in the United States, Germany, and Japan [38]. Afterwards the mass cultivation of algae became one of the hottest topics in algal biotechnology leading to the development of diverse culture systems for mass culture applications [39–42]. Generally, algal cultures can be grown photoautotrophically, heterotrophically, or mixotrophically, in open or closed culture systems.

4.1 Photoautotrophy

Chlorella possesses high PE and is commonly cultured outdoors driven by the sunlight in open ponds or closed photobioreactors (PBRs). The popularly used open ponds include circular ponds and raceway ponds. Circular ponds were first built in Japan and then introduced to China and are now widely employed for mass cultivation of Chlorella in Asia. The capacity scale-up of circular ponds can be achieved by increasing the pond diameter, which can reach up to 50 m [40]. The system has several disadvantages including requirement of expensive structures of heavy reinforced concrete, high energy consumption for continuous stirring, inefficiency in land use, and so on. The raceway pond is another popular open culture system in the world. The raceway pond was initially used for commercial production of Spirulina and is now also employed for mass culture of Chlorella. The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid. A raceway pond appears to be a single unit or in the form of a meandering channel assembled by individual raceways [39]. Generally, the cultures in open ponds are kept shallow at a depth of no more than 30 cm to facilitate the penetration of sunlight. Overall, the cell density achieved in both circular and raceway ponds is relatively low and commonly less than 1 g dry weight L−1. In contrast, the cascade system, a newly developed open culture system by Czech researchers, is capable of reaching a high cell density of up to 40 g L−1 [41, 43, 44]. This system is characterized by the thin layer of suspension (ca. 6 mm), highly turbulent flow, and high ratio of exposed surface area to total volume. Although the cascade system can achieve high volumetric cell density, its overall areal productivity is just comparable to that of open ponds [43].

Open systems cost less to construct and maintain and are thus regarded more economically favorable than closed PBRs, but they have substantial intrinsic disadvantages including low cell density and biomass productivity, rapid water loss due to evaporation, easy contamination by other microorganisms, and difficulty in managing culture temperature and efficient CO2 delivery. The ease of contamination is a common problem encountered in outdoor cultures. Chlorella culture in open ponds is susceptible to other unwanted algae including diatoms and cyanobacteria, and protozoans such as rotifers, ciliates, and amoebae that feed on the algae, resulting in greatly reduced production of Chlorella biomass. Developing a best management practices plan for prevention and treatment of contamination may represent a feasible approach toward increased production economics of Chlorella by open systems.

Closed PBRs have the potential to overcome the problems of evaporation, contamination, and low biomass productivity encountered in open systems. PBRs are made of transparent materials with a large ratio of surface area to volume. Tubular PBR is one of the most popular designs. It can be arranged as straight tubes horizontally placed parallel to each other, α-type cross tubes at an angle with the horizon, or coiled tubes helically surrounding a supporting frame [39, 45–47]. Schenk et al. [47] reported a large tubular PBR system installed in Germany, which consists of 500 km of tubes arranged as fences in a north/south direction. This system has a capacity of 700 cm3 with the annual production of Chlorella biomass up to 100 t. Panel PBR is another popular design arranged either vertically or inclined to the ground [48–51]. Compared with tubular PBR, the panel design needs less capital for construction and operation, offers less dark volume, accumulates less dissolved oxygen, and so on. To date, the Arizona Center for Algae Technology and Innovation (AzCATI) at Arizona State University (United States) has launched a 0.5 acre of a panel PBR system for investigating the biomass production potential of Chlorella cultures.

4.2 Mixotrophy

Many Chlorella strains are able to grow robustly under mixotrophic conditions utilizing both CO2 and organic carbons in the presence of light (Table 1). Usually, Chlorella grows better under mixotrophic conditions than under autotrophic conditions [32, 36, 52]. Therefore, in some cases, organic carbons are added into open ponds or PBRs to achieve mixotrophic production of Chlorella biomass [40, 53]. However, the Chlorella cultures in open ponds supplemented with organic carbons, sugars in particular, are highly susceptible to bacterial contamination. To reduce the chance of contamination, stepwise feeding of acetate but not sugars is employed. Inasmuch as the PBR system is closed, it offers better performance than an open system to maintain a monoculture under mixotrophic conditions. There was a report of successful maintenance of Chlorella monoculture mixotrophically grown in outdoor PBRs supplemented with sugars [54]. But the monoculture was achieved only in small-volume PBRs (i.e., 10 L) and it got contaminated and crashed when the culture volume scaled up to 300 L. The fermenter system that was conventionally used for fermentation of nonphotosynthetic organisms such as bacteria, yeasts, and animal cells, has also been proposed to mixotrophically grow Chlorella by providing sugars and artificial light sources [55]. The fermenter design, however, is less efficient in light usage and mixotrophic growth, particularly when using large-volume fermenters for high density of cultures.

4.3 Heterotrophy

Heterotrophic growth of Chlorella in fermenters has a long history and is now gaining increasing attention [30, 32, 40, 56, 57]. Fermenters are usually placed indoors without provision of light. Almost all Chlorella species reported are able to grow robustly under heterotrophic conditions with the addition of organic carbon sources, sugars in particular (Table 1). These organic carbon sources, including sugars, hydrolyzed carbohydrates, acetate, and glycerol, serve as the solo carbon and energy sources to support the growth of Chlorella. Chlorella protothecoides and Chlorella zofingiensis are the most well-studied Chlorella species for fermentation. Li et al. [58] reported a scale-up of heterotrophic production of C. protothecoides in an 11,000-L fermenter with the cell density reaching up to 13 g L−1, which is comparable to that achieved in a 5-L fermenter. The competitiveness of heterotrophic production of Chlorella over photoautotrophic production rests largely with high cell density and great biomass productivity, elimination of the light requirement, ease of control for monocultures, and low-cost biomass harvesting [59]. The high cell density and biomass productivity of heterotrophic Chlorella can be achieved by the employment of fed-batch, continuous, and cell-recycle culture strategies that are well developed for the fermentation of bacteria or yeasts [31, 32, 56, 60, 61]. With the optimized fermentation conditions, heterotrophic Chlorella was reported to achieve as high as 100 g cell dry weight per liter with an average biomass productivity of 13 g L−1 day−1 [61]. Although Chlorella fermentation has been gaining the increasing attention of industry, it is regarded economically favorable only for high-value products but not for the low-cost commodity products such as biofuels, because of the relatively high production cost.

Each culture strategy mentioned above has its own advantages and disadvantages. The choice of cultivation methods depends on Chlorella species/strains, locations of culture systems, production capacities, desired products, and so on. In some cases, hybrid systems (e.g., PBR-pond and fermenter-PBR) instead of a sole culture system can be employed. In a PBR-pond system, Chlorella cells are cultured in PBRs for rapid growth, which serve as the inoculation seed in raceway ponds for large-scale biomass production. In a fermenter-PBR system, Chlorella cells are first grown heterotrophically in fermenters for accumulation of high-density biomass, which are then transferred to thin PBRs with high light for induction of desired products, for example, oils or astaxanthin. Regardless of algal strains and culture systems, the key to optimizing a production system lies in the cost balance of output to input.

5 Potential Applications

Chlorella is the sunlight-driven single-cell factory for protein, lipids, carbohydrates, pigments, vitamins, and minerals. It has long been used as health food and additives for human consumption, as well as animal feed in aquaculture. In addition, the green alga proves to be beneficial to environmental cleanup such as bioremediation of industrial flue gases and wastewater. Recently, due to the blooming of renewable energy, Chlorella has attracted unprecedented interest as a feedstock for biofuels, biodiesel in particular. Although the microalgal expression system does not reach a stage as mature as bacteria, yeast, mammalian cell, or plant systems, it shows substantial advantages and has been used increasingly for expression of recombinant proteins.

5.1 Chlorella as Human Food and Animal Feed

Chlorella is abundant in protein (up to 68 %) and contains all the essential amino acids. It is also rich in fatty acids, dietary fibers, carotenoids, vitamins, minerals, and other bioactive compounds, enabling the alga an attractive foodstuff of high nutritional quality. The use of Chlorella as nutritional food has a long history and can be traced back to food shortage periods during the World Wars. Japan and China are the main Chlorella-producing countries, with an annual production of over 3,500 t of biomass in 2005 [40]. Yaeyama Chlorella (Japan), Sun Chlorella (Japan), and Taiwan Chlorella are the most popular companies for Chlorella production. The produced Chlorella is commercialized mainly in the form of dried powder, tablets, or capsules for human consumption. Other forms of products include Chlorella growth factor (CGF), Chlorella tea, Chlorella noodles, and the like. CGF is a hot-water extract of Chlorella and represents a mixture of proteins, nucleic acids, polysaccharides, and a variety of minerals [40]. Administration of Chlorella or Chlorella extracts has been shown to play positive roles in health care and disease prevention, such as boosting immune functions [62, 63], preventing tumors and cancers [64, 65], enhancing hypoglycemic effects [66, 67], attenuating cognitive decline in age-dependent dementia [68], and lowering blood pressure [69]. Chlorella is also used as a natural color additive for human food because of its high levels of pigments [70, 71]. As stated by [70], the addition of Chlorella biomass gave cookies an attractive and innovative appearance and higher textural characteristics.

The use of Chlorella as animal feed is more recent. Nutritional Chlorella biomass can be used directly as feed or to enrich protozoa such as rotifers that serve as feed in aquaculture [72, 73]. Feeding of Chlorella proves beneficial to the growth and nutritional improvement of fish. The level of skin pigmentation is one of the most important quality criteria determining the market value of fish, ornamental fish in particular. They are unable to synthesize carotenoids de novo and have to feed on the carotenoid-containing organisms (e.g., microalgae) to achieve their natural pigmentation. Chlorella is rich in pigments and even keto-carotenoids depending on species and therefore is popularly used in aquaculture for coloring ornamental fish [74–76]. In addition, Chlorella shows promising applications in poultry, for example, feeding to hens to color their egg yolks [77]. Because Chlorella has a tough rigid cell wall, proper pretreatment is commonly necessary to facilitate the digestion and assimilation of nutrients from Chlorella [78, 79].

5.2 Chlorella as a Source of Carotenoids

Carotenoids commonly found in Chlorella include α- and β-carotenes, lutein, zeaxanthin, violaxanthin, and neoxanthin. Some keto-carotenoids such as canthaxanthin and astaxanthin are also found in certain Chlorella species [33]. Figure 1 shows the schematic pathway of carotenoid biosynthesis in Chlorella. Generally, hydroxylation of the C-3 and C-3′ positions of β-carotene and α-carotene results in the formation of zeaxanthin and lutein via β-cryptoxanthin and α-cryptoxanthin, respectively. The subsequent epoxidation of zeaxanthin leads to the production of violaxanthin which is further converted to neoxanthin. In Chlorella zofingiensis additional keto-carotenoid biosynthetic pathways are present, involving several oxygenation and hydroxylation steps that lead to the formation of astaxanthin from β-carotene [80]. Carotenoids have important applications in food, feed, nutraceutical, and pharmaceutical industries because of their strong coloring ability, powerful antioxidative activity, and beneficial effects on human health [81]. Using Chlorella as producers of lutein and astaxanthin has been proposed [31, 32, 82].

Fig. 1
figure 1

Schematic diagram of carotenoid biosynthesis in Chlorella. In the box is the astaxanthin biosynthetic pathway which is present in Chlorella zofingiensis. IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, GGPP geranylgeranyl pyrophosphate, PSY phytoene synthase, PDS phytoene desaturase, ZDS ζ-carotene desaturase, CRTISO carotene isomerase, LCYb lycopene β-cyclase, LCYe lycopene ε-cyclase, P450b-CHY cytochrome P450 β-hydroxylase, P450e-CHY cytochromeP450 ε-hydroxylase, CHYb β-carotene hydroxylase, BKT β-carotene ketolase, ZEP zeaxanthin epoxidase, VDE violaxanthin de-epoxidase, NXS neoxanthin synthase, AAT astaxanthin acyltransferase

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Shi et al. [83] analyzed seven Chlorella strains for lutein production and the results suggested that Chlorella protothecoides CS-41 was a potential producer of lutein inasmuch as it accumulated the highest level of lutein (4.5 mg g−1 dry weight). Later, Shi and Chen [82] investigated the growth and lutein production of C. protothecoides under both heterotrophic and mixotrophic culture conditions; mixotrophic cultures produced more biomass and higher amounts of lutein than heterotrophic ones. It was revealed that C. protothecoides was able to utilize various nitrogen sources including nitrate, ammonia, and urea, but urea proved to be superior to the other two nitrogen sources for growth and lutein production [24]. In order to increase lutein production, Shi and Chen [84] adopted a fed-batch culture strategy to grow C. protothecoides with urea as the nitrogen source, achieving a lutein yield up to 225 mg L−1 with the maximal productivity of 48 mg L−1 day−1. The comparable lutein productivity was obtained when the fed-batch process was scaled up to a 30-L culture volume [84], indicating the potential of using heterotrophic C. protothecoides for scalable production of lutein.

C. zofingiensis is the only Chlorella species known to synthesize keto-carotenoids including astaxanthin. The astaxanthin production potential of C. zofingiensis has been studied under photoautotrophic, heterotrophic, and mixotrophic conditions [33, 55, 85, 86]. Under photoautotrophic conditions, stresses such as high light intensity and/or nitrogen starvation are needed to induce astaxanthin accumulation in C. zofinginesis [87]. These stresses, however, are unfavorable for algal growth and biomass production. In addition, the attenuated light absorption caused by mutual shading of cells severely affects the productivity and quality of algal biomass and products. In contrast, heterotrophic cultivation of C. zofingiensis feeding on an organic carbon source can boost algal growth as well as astaxanthin accumulation, eliminating the need for light and light-associated growth issues [33]. It has been reported that heterotrophic cultures of C. zofingiensis could achieve a comparable astaxanthin yield to H. pluvialis on a volumetric basis, much higher than that under photoautotrophic conditions [87, 88]. In this context, heterotrophic culture mode is regarded to be more feasible than autotrophic mode for astaxanthin production by C. zofingiensis. Sugars, glucose in particular, are commonly used for heterotrophic production of astaxanthin from C. zofingiensis, making the production relatively expensive and thus hampering its commercial application to some extent. To reduce the production cost, waste sugars such as cane molasses have been proposed to replace glucose for astaxanthin accumulation [31, 32]. Cane molasses is a by-product of the sugar industry consisting mainly of sucrose, glucose, and fructose, and is much cheaper than glucose. It has been proved that molasses gave a comparable astaxanthin productivity to glucose [32], opening up a possibility of using industrially cheap organic carbons toward large-scale and cost-saving production of astaxanthin. Although C. zofingiensis can achieve high cell density under heterotrophic conditions, the intracellular astaxanthin content is relatively low compared to that under phototrophic conditions with high light and nitrogen starvation, offsetting in part the production economics of astaxanthin.

We have developed a heterotrophic–phototrophic two-stage culture strategy to grow C. zofingiensis to improve astaxanthin production: C. zofingiensis was first cultured in the presence of glucose in the dark for rapid accumulation of biomass, which was then transferred to high light conditions for induction of astaxanthin. The new culture strategy greatly enhanced the intracellular accumulation of astaxanthin to 3.5 mg g−1 of dry weight, which is 3.2 times the astaxanthin content obtained under heterotrophic conditions (unpublished data). The drastic increase in astaxanthin content may lie in that the alga needs more astaxanthin to cope with the light-associated adverse effects. Additionally, a record high astaxanthin productivity of 4.7 mg L−1 day−1 was achieved, which is 2.9- and 2.4-fold higher than that under heterotrophic and phototrophic conditions, respectively (unpublished data). The newly developed heterotrophic–phototrophic two-stage culture strategy combines the advantages of both heterotrophic and phototrophic modes and eliminates the possible contamination associated with mixotrophic growth, which can significantly enhance astaxanthin production and may open up the possibility of substituting the currently used heterotrophic culture method for commercial production of astaxanthin at large scale.

Another strategy to overcome the low astaxanthin content is to manipulate the carotenoid biosynthetic pathway in C. zofingiensis through genetic engineering. Aside from astaxanthin, C. zofingiensis contains substantial amounts of canthaxanthin and adonixanthin [89], suggesting that CHYb may not accept canthaxanthin as a substrate to produce astaxanthin and BKT might be insufficient to catalyze the formation of astaxanthin from adonixanthin in C. zofingiensis. It has been reported that CHYb from H. pluvialis can utilize canthaxanthin as the substrate for efficient synthesis of astaxanthin [90] and BKT from C. reinhardtii has a high activity of converting adonixanthin to astaxanthin [91]. Therefore, the manipulation of specific astaxanthin biosynthetic steps by introducing these two genes into C. zofingiensis may provide a pulling force for astaxanthin synthesis at the cost of both canthaxanthin and adonixanthin, which, when coupled with the pushing force from PDS overexpression, may represent a feasible strategy to increase astaxanthin content and purity further. We have developed a sophisticated transformation system for C. zofingiensis [80], whereby the genetic engineering for astaxanthin enhancement is possible.

5.3 Chlorella for CO2 Biomitigation and Wastewater Bioremediation

Global warming caused by the increasing greenhouse gases in the atmosphere has attracted great concern by the public and scientific community. Carbon dioxide is the principal greenhouse gas mainly released through burning of fossil fuels. The biomitigation of CO2 by autotrophs such as microalgae is a promising strategy proposed for fixing CO2 and attenuating the greenhouse effect. Chlorella is one of the most commonly used genera of algae for sequestration of CO2 due to its high growth rate and strong CO2 fixation ability [19, 20, 92–94]. Chlorella is able to fix CO2 from different sources, which can be simply classified as air, CO2-enriched air, and industrial exhaust gases such as flue gas. The CO2 fixation rate is associated with the CO2 concentration provided for Chlorella growth (Fig. 2). When provided with atmospheric air (ca. 0.04 % CO2), Chlorella grows poorly and shows low fixation abilities due to the mass transfer limitation. Therefore, to facilitate algal growth, the CO2- enriched air at the concentration of 1–5 % is usually provided, accompanied by the increased CO2 fixation rate. However, when the CO2 content is over 5 %, the fixation rate drops down significantly (Fig. 2).This may be mainly attributed to the decreased medium pH at high CO2 concentration that inhibits the growth of Chlorella. The flue gases contain up to 15 % CO2 and are responsible for more than 7 % of the total world CO2 emissions [95]. It will be environmentally and cost beneficial if Chlorella can directly use flue gases for CO2 fixation, which requires strains tolerant to high CO2 concentration as well as to relatively high temperature. High-CO2 tolerant Chlorella strains have been reported, some of which were able to grow in the presence of up to 40 % CO2 at 42 °C without significant growth inhibitory effects [22, 96].

Fig. 2
figure 2

Relative CO2 fixation rate of Chlorella as affected by CO2 concentration. Data are based on studies during the past five years

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The tolerance to both high CO2 and high temperature enables these Chlorella strains to be potential cellular reactors for the biomitigation of CO2 from flue gases. For example, Chlorella sp. UK001, one of the CO2 tolerant strains, showed a CO2 fixation rate of more than 1 g L−1 day−1 when aerated with 15 % CO2 [97], much higher than that of regular Chlorella strains. In addition, the choice of culture system affects the capacity of CO2 fixation. Doucha et al. [98] reported the CO2 fixation of flue gas from a natural gas-fired boiler by Chlorella cultivated in an outdoor open cascade system with a culture area of 55 m2. The inhibition of algal growth caused by sulphur and nitrogen oxides (SOx, NOx) that exists in flue gas [99] was not observed in this study. The biomass productivity and PAR utilization of Chlorella cultures saturated with flue gas were 19.4–22.8 g m−2 day−1 and 5.58–6.94 % respectively, comparable to that with pure CO2. Later, Douskova and Livansky [100] investigated the CO2 fixation rate by Chlorella vulgaris in aerated columns with flue gas or CO2-enriched air. Flue gas-aerated Chlorella cultures exhibited an even higher CO2 fixation rate (4.4 g L−1 day−1) than that aerated with CO2-enriched air (3.0 g L−1 day−1). Recently, there have been increasing reports of using Chlorella wild-type or mutant strains to sequestrate CO2 from industrial flue gas [92, 101–103].

It is noteworthy that a high concentration of CO2 generally leads to low efficiency of CO2 removal; for example, the removal efficiency by Chlorella sp. in the presence of 15 % CO2 is only 16 %, much lower than that in the presence of 2 % CO2 (58 %, [19]), indicating that a major portion of CO2 is released from the cultures. This can be overcome in part by passing the gases through sequential culture units where CO2 is resequestrated toward less emission. For example, the fixation efficiency of flue gas CO2 achieved by Chlorella cultures in sequential bioreactors reaches up to 85.6 %, greatly higher than that obtained in a single bioreactor [104]. As suggested by Doucha et al. [98], the daily fixed CO2 per m2 is around 34.4 g, with the simultaneous production of 20 g algal biomass. Assuming the culture season lasts for 150 days, one hectare of Chlorella cultures is able to sequestrate 21 t CO2 and produce around 12 t biomass per year.

Bioremediation using microalgae has long been recognized as an environmentally sound approach for wastewater treatment. Chlorella is one of the microalgal genera widely used in the biological treatment of wastewater and has proven abilities of removing nutrients (N and P), organic contaminants, and heavy metals (Table 2). Generally, pretreatments of wastewater such as settling, activated sludge process, or dilution are needed before supplying to Chlorella for biological treatment [105–108]. Wang et al. [109] intensively investigated the growth of Chlorella sp. on wastewater sampled from four different points of the treatment process flow of a municipal wastewater treatment plant for the removal of nitrogen, phosphorus, and chemical oxygen demand (COD) as well as metal ions. The four types of wastewater are classified as wastewater before primary settling (#1), wastewater after primary settling (#2), wastewater after activated sludge tank (#3), and concentrate (#4). The growth rate (0.95 day−1) and COD removal rate (83.0 %) of Chlorella for wastewater #4 were much higher than those for wastewater #1 and #2 and the removal rates of nitrogen (78.3 %) and phosphorus (85.6 %) were comparable; Chlorella in wastewater #3 showed the lowest growth rate and removal rates of nitrogen, phosphorus, and COD. The efficient removal of nutrients and organic contaminants by Chlorella from wastewater was also demonstrated in other studies [107, 110–114]. These results suggest that growing Chlorella on wastewater seems to be a feasible strategy to reduce the released amounts of organic and inorganic nutrients into natural waters, thus preventing the eutrophication problem.

Table 2 Selected Chlorella species reported for wastewater treatment
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Although the suspended Chlorella cultures exhibited their potential use in secondary or tertiary steps for wastewater treatment, one of the major and practical limitations is separation of the algal biomass from the treated wastewater, which requires capital-intensive steps such as flocculation, flotation, filtration, or centrifugation. In this context, using immobilized Chlorella cells for wastewater treatment is advantageous in that no harvest step is required [106, 115–117]. The removal efficiency of nutrients by immobilized Chlorella is influenced by culture density, pH, and immobilizing matrix [115, 118]. In some cases, Chlorella was cocultured with other microalgae for wastewater treatment [113]. In addition to removal of nutrients and organic compounds, Chlorella is also able to be used for the biodegradation of toxics [119] and removal of metal ions [109, 115].

The biosorption of Chlorella for removing metals from wastewater involves adsorption of metal ions onto the cell surface and binding to the intracellular molecules such as cytoplasmic ligands, phytochelatins, and metallothioneins (for details see the review by Mehta and Gaur [120]). Chlorella has been reported to remove a wide range of metals, including Al, Ca, Cd, Cu, Fe, Mg, Mn, Ni, Ur, and Zn [109, 121–125]. Considering the acceptable growth and lipid production of Chlorella on wastewater, the integration of biofuel production with wastewater treatment is proposed [109, 111, 112, 114, 126].

5.4 Chlorella as Feedstock for Biofuels

Petroleum fuels are recognized to be unsustainable due to their depleting supplies and release of greenhouse gas [127]. Renewable biofuels are promising alternatives to petroleum, among which biodiesel has attracted unprecedentedly increasing attention in recent years [128]. Compared with traditional fuels, the carbon-neutral biodiesel releases fewer gaseous pollutants and is considered environmentally beneficial. Currently, biodiesel is produced mainly from vegetable oils, animal fats, and waste cooking oils. Plant-oil–derived biodiesel, however, cannot realistically meet the existing need for transport fuels as immense arable lands have to be occupied in cultivating oil crops, causing food–fuels conflicts [39].

Microalgae have been considered as the promising alternative feedstock for biodiesel production because of their rapid growth and high oil content [39, 128, 129]. Furthermore, unlike oil crops, microalgae can be easily cultured in outdoor ponds or bioreactors, making them superior to oil crops in biomass production. Chlorella represents a group of green microalgal species that grow fast and are easily able to achieve and maintain mass cultures. There have been many research studies focusing on using Chlorella for biodiesel production in the past years, as shown in Table 3. Under optimal conditions Chlorella usually synthesizes a relatively low content of lipids (25 % on average) which can be greatly increased up to 66 % by stress conditions such as nitrogen starvation [17]. The stress conditions also favor the accumulation of neutral lipids, in particular, triacylglycerols (TAGs) that deposit in the cytosol. TAGs are considered to be superior to polar lipids (phospholipids and glycolipids) for biodiesel production. In this context, Chlorella can be first cultured under favorable conditions to maximize biomass production and then exposed to stress conditions to stimulate the accumulation of lipids including TAGs. The important properties of biodiesel such as cetane number, viscosity, cold flow, and oxidative stability are largely determined by the composition and structure of fatty acyl esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstock, for example, carbon chain length and unsaturation degree [130]. The synthesized fatty acids in Chlorella are mainly of medium length, ranging from 16 to 18 carbons, despite the great variation in fatty acid composition (Table 4). Generally, saturated fatty esters possess a high cetane number and superior oxidative stability whereas unsaturated, especially polyunsaturated, fatty esters have improved low-temperature properties [131]. It is suggested that the modification of fatty esters, for example, enhancing the proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote the quality of biodiesel [132]. In this regard, C. protothecoides, which has the highest proportion of oleic acid (71.6 %), may be better than other Chlorella species as biodiesel feedstock [34]. The properties of C. protothecoides derived biodiesel were assessed and most of them proved to comply with the limits established by American Society for Testing and Materials (ASTM), including density, viscosity, flash point, cold filter plugging point, and acid value.

Table 3 Selected Chlorella species reported for biodiesel production research
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Table 4 Fatty acid profiles of selected Chlorella species
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Aside from employing photoautotrophic Chlorella cells, fermentation by feeding organic carbons has also been proposed by some research groups to enrich heterotrophic biomass as biodiesel feedstock. C. protothecoides is the most studied species heterotrophically grown for biodiesel production (Table 5). It could achieve very high cell densities, biomass productivities, and lipid productivities. Glucose is the most widely used carbon source to feed Chlorella for boosting biomass production and lipid accumulation. To reduce the production cost, alternative low-cost carbon sources such as hydrolysates of crude carbohydrates, waste molasses, or glycerol were used [34, 35, 61, 133–136]. Molasses proved to be a promising alternative to feed C. protothecoides for biodiesel, with the biomass yield, biomass productivity, and lipid productivity being 97.1 g L−1, 12.8, and 7.3 g L−1 day−1, respectively [61]. However, the conversion ratio of sugar to biomass in these heterotrophic cultures was restricted to 0.5, which means 2 t of sugar are required for producing 1 t of biomass and 1 t of CO2 is released during this process. In this regard, fermentation is neither economically viable nor environmentally friendly for the production of biomass for biodiesel as compared with photoautotrophy.

Table 5 Growth and lipid production of C. protothecoides feeding on various organic carbon sources
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Transesterification is needed to convert Chlorella oil to biodiesel. It is a chemical conversion process involving reacting triglycerides catalytically with a short-chain alcohol (typically methanol or ethanol) to form fatty acyl esters (biodiesel) and glycerol. This reaction occurs stepwise with the first conversion of triglycerides to diglycerides and then to monoglycerides and finally to glycerol. Considering the reaction is reversible, a large excess of alcohol is used in industrial processes to ensure the direction of fatty acid esters. Methanol is the preferred alcohol for industrial use because of its low cost, although other alcohols including ethanol, propanol, and butanol are also commonly used. In addition to heat, a catalyst is needed to facilitate the transesterification. The transesterification of triglycerides can be catalyzed by acids, alkalis, or enzymes [137–139]. Currently, alkali (sodium hydroxide and potassium hydroxide) is the preferred type of catalyst for industrial production of biodiesel.

Chlorella is also capable of producing other biofuels such as hydrogen, ethanol, methane, and biocrude [140–144]. The biohydrogen production from microalgae has long been recognized. This process involves hydrogenase, an enzyme highly sensitive to O2. During photosynthesis, hydrogen evolution is transient due to the strong inhibition of hydrogenase by photosynthetically evolved O2 [145]. The sustainable hydrogen production from Chlorella, therefore, requires the maintenance of an anaerobic environment, which can be achieved through the inhibition of O2 evolution by sulfur deficiency [146]. It has been estimated that green algae could theoretically produce a maximum of 20 g hydrogen per m2 per day [145]. In addition to lipids, Chlorella biomass contains a substantial amount of carbohydrates (starch and cellulose) that can be used for ethanol production through technologies such as saccharification and fermentation [142, 147, 148]. Chlorella is also capable of producing ethanol through self-fermentation of intracellular starch under dark and anaerobic conditions, although the conversion efficiency is relatively low [142]. Anaerobic digestion of biomass results in the production of methane that can be used as a heat source or for electricity generation. The raw Chlorella biomass can be directly subjected to digestion, thus avoiding the biomass-harvest and oil-extraction processes used in algal biodiesel production and significantly bringing down the production cost and energy debt. But the digestion efficiency as stated by Ras et al. [143] was restricted to 50 %, indicating the need of proper pretreatments of the raw biomass for complete digestion. Hydrothermal liquefaction, which requires moderate temperatures as compared to the processes of pyrolysis and gasification, is commonly used for biocrude production from Chlorella biomass [141, 149].

The production of biofuels from microalgae is still far from commercialization. There are significant technical challenges yet to be addressed. A promising strategy is to integrate the production of biodiesel with other biofuels and high-value products, as well as the applications of treating flue gas and wastewater (Fig. 3).

Fig. 3
figure 3

Schematic illustration of integrated production of biofuels and other products coupled with flue gas and wastewater treatments

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5.5 Chlorella as Cell Factories for Recombinant Proteins

Chlorella offers substantial advantages as a promising alternative to currently well-established expression systems of bacteria, yeast, and mammalian cells. In contrast to bacteria, Chlorella belongs to eukaryotic organisms and therefore can perform correct posttranscriptional and posttranslational modifications essential for the production of functional eukaryotic proteins. Being easy, rapid, and inexpensive to grow and maintain on a large scale both indoors and outdoors, Chlorella is more cost effective as compared with the capital-intensive and time-consuming expression systems of mammalian cells, large farm animals, and higher plants. In addition, Chlorella has long been approved and used as health food for human consumption, suggesting the biological safety of engineered proteins from Chlorella. These characteristics enable Chlorella to be potentially useful as a bioreactor for synthesizing engineered products of interest such as enzymes, vaccines, monoclonal antibodies, and growth factors.

Nevertheless, the utilization of Chlorella for heterologous expression is hampered by the lack of a sophisticated genetic toolbox. The genetic engineering of Chlorella has achieved some success during the past 20 years (Table 6). The first report was conducted by Jarvis and Brown [150] who introduced a firefly luciferase gene into Chlorella ellipsoidea for heterologous expression; the luciferase activity, however, was not stable and disappeared after a few days. Later, Maruyama et al. [151] performed the transient expression of the β-glucoronidase (GUS) gene in transgenic Chlorella saccharophila. The transient expression of GUS was also reported in transgenic Chlorella ellipsoidea [152, 153] and Chlorella sp. [154]. These results suggested the feasibility of using Chlorella as an expression system for recombinant protein production. A dominant selectable marker is essential for easy and reliable selection of target Chlorella transformants. The frequently used selectable markers include bleomycin binding protein (Ble, resistant to phleomycin), chloramphenicol acetyltransferase (Cat, resistant to chloramphenicol), hygromycin B phosphotransferase (Hpt, resistant to hygromycin), and neomycin phosphotransferase II (NptII, resistant to kanamycin and geneticin), derived from either Escherichia coli or Streptoalloteichus rimosus (Table 6). Chlorella, however, is not so sensitive to these antibiotics and may need high concentrations to inhibit its growth completely [155]. In addition, Chlorella harboring these bacterial genes may be subject to biological safety problems when used as food or pharmaceuticals for human beings. Therefore, endogenous genes are advantageous as selectable markers for Chlorella transformation. Dawson et al. [156] reported the use of the nitrate reductase (NR) gene from Chlorella vulgaris to rescue the NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants. Wang et al. [157] also reported the stable transformation of Chlorella ellipsoidea using an endogenous nitrate reductase gene to complement the NR-deficient mutant. The limitation of these trials lies in that a NR-deficient mutant is required as the expression host. Very recently, a phytoene desaturase (PDS) from a norflurazon-resistant mutant of Chlorella zofingiensis was proposed as a dominant selectable marker for stable transformation of Chlorella [80, 89, 158]. The Chlorella mutant harbored a point mutation on its PDS, which showed great resistance to norflurazon as well as significantly enhanced desaturation activity. It has been demonstrated that as low as 0.25 μg mL−1 of norflurazon is sufficient to select target transformants of Chlorella zofingiensis transformed with the mutated PDS gene. The transformants retained the norflurazon resistance after more than 100 times of subculture without selection of norflurazon, suggesting the great potential of using the endogenously derived PDS gene mutant as an effective and efficient selectable marker for the stable transformation of Chlorella zofingiensis as well as other Chlorella species.

Table 6 Summary of Chlorella species for genetic modification
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Genetic manipulation of Chlorella for heterologous gene expression is still in its infant stage. The introduced foreign genes are subject to instability if not lack of expression because of various possible reasons including unstable nuclear integration, position effects, inefficient transcription from heterologous promoters, inaccurate RNA processing, and codon usage bias. Nevertheless, there have been several reports of using Chlorella for expressing commercially interesting proteins such as rabbit neutrophil peptide-1 [159–161], human growth hormone [155], flounder growth hormone [162], mercuric reductase from Bacillus megaterium [163], and trypsin-modulating oostatic factor from mosquitos [164]. Challenges for getting stable transgene integration and expression in Chlorella transformants will require further investigations to develop sophisticated genetic toolboxes with a powerful expression cassette for this small-sized and cell-wall–tough species.

6 Conclusions and Future Prospects

Chlorella is a sunlight-driven single-cell bioreactor that converts carbon dioxide to potential proteins, lipids, carbohydrates, and high-value biocompounds. It is among the most well-studied genera of microalgae for mass cultures. The abundance in protein and other nutritional elements, biological safety, and feasibility of growing and maintaining outdoors on a large scale enable Chlorella to be a good source of health food for human consumption. Chlorella is also considered as a potential source of microalgal oils for biofuel production. There are still substantial challenges involved in the biofuel production pipeline such as mass cultivation, harvest and drying, biomass disruption for oil extraction and conversion, and recycling of water and nutrients, making the Chlorella-derived biofuels currently capital intensive and far from economically viable as compared with fossil fuels. The integrated production of biofuels and other potential high-value products, coupled with the environmentally beneficial applications such as flue gas biomitigation and wastewater treatment represent a promising direction toward a cost-effective production process of Chlorella, which requires close collaboration between biologists and engineers. As interest in Chlorella increases, comprehensive analyses of certain potential strains are underway via genomic, transcriptomic, proteomic, lipidomic, and metabolomic approaches. The availability of those omics data will uncover the biological implications and facilitate the tailored manipulation of Chlorella for broader industrial applications.