Mixotrophic cultivation of microalgae for biodiesel production: status and prospects.

Appl Biochem Biotechnol DOI 10.1007/s12010-014-0729-1

Mixotrophic Cultivation of Microalgae for Biodiesel Production: Status and Prospects Jinghan Wang & Haizhen Yang & Feng Wang

Received: 21 March 2013 / Accepted: 2 January 2014 # Springer Science+Business Media New York 2014

Abstract Biodiesel from microalgae provides a promising alternative for biofuel production. Microalgae can be produced under three major cultivation modes, namely photoautotrophic cultivation, heterotrophic cultivation, and mixotrophic cultivation. Potentials and practices of biodiesel production from microalgae have been demonstrated mostly focusing on photoautotrophic cultivation; mixotrophic cultivation of microalgae for biodiesel production has rarely been reviewed. This paper summarizes the mechanisms and virtues of mixotrophic microalgae cultivation through comparison with other major cultivation modes. Influencing factors of microalgal biodiesel production under mixotrophic cultivation are presented, development of combining microalgal biodiesel production with wastewater treatment is especially reviewed, and bottlenecks and strategies for future commercial production are also identified. Keywords Microalgae . Mixotrophic cultivation . Cultivation mode . Biodiesel . Wastewater treatment . Lipid

Introduction Biodiesel is now considered as a promising source for alternative fuel production owing to its non-toxic, clean, biodegradable, and renewable characteristics [1–3]. As a newly emerged source of biodiesel, microalgal biodiesel has much higher productivity than traditional biodiesel from energy crops due to short growth cycles [4–8]. Besides, microalgae can be cultivated with brackish or wastewater on non-arable land for year-round production, which further relieves the stress on arable land, freshwater, and food production [9–11]. Figure 1 displays a flow chart of producing biodiesel from microalgae. The solid lines describe the basic steps; the dotted lines show the factors that should be taken into consideration to maximize the products of interest. J. Wang : H. Yang : F. Wang (*) Research Institute of Environmental Planning and Management, College of Environmental Science & Engineering, Tongji University, Shanghai 200092, China e-mail: [email protected] J. Wang : H. Yang : F. Wang State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science & Engineering, Tongji University, Shanghai 200092, China

Appl Biochem Biotechnol

Fig. 1 A flow chart of producing biodiesel from microalgae

To achieve the largest possible microalgal biodiesel productivity in a cost-effective way, selection of microalgae cultivation modes is of vital importance. Three major modes of cultivation can be adopted for microalgal biodiesel production, namely photoautotrophic, heterotrophic, and mixotrophic cultivation. As a newly adopted yet effective mode for producing microalgal biodiesel, the status and prospects of producing microalgal biodiesel under mixotrophic cultivation have not been fully recognized. The objectives of this paper are to elaborate the mechanisms and virtues of mixotrophic cultivation through comparison with the other two modes, to summarize influencing factors of mixotrophic cultivation of microalgae for biodiesel production, and to review the development of combining microalgal biodiesel production with wastewater treatment. Bottlenecks and strategies for future development are also identified.

Mechanisms of Mixotrophic Cultivation The mode of cultivation influences significantly the growth pattern of microalgae, thus further determines the quality and quantity of biodiesel products. Mechanisms and characteristics of the three major cultivation modes, i.e., photoautotrophic, heterotrophic, and mixotrophic cultivation, are discussed in the following section. Photoautotrophic Cultivation Photoautotrophic cultivation is the most commonly employed and most energy-saving mode of microalgae cultivation, generally carried out in open ponds or photobioreactors [12–16]. Under this mode, chemical energy is formed through photosynthesis, in which process


microalgae utilize light as the energy source and inorganic carbon as the carbon source. A CO2-rich environment could enhance biomass productivity to a certain extent [17, 18]; however, since light penetration decreases exponentially with the increase of broth turbidity (caused mainly by microalgal cells), photoautotrophic cultivation has difficulty achieving high biomass concentration and biomass productivity after all [19]. Lipid content and lipid productivity are parameters of great importance for biodiesel production [20, 21]. The lipid content (dry weight of biomass) of microalgae varies widely under photoautotrophic cultivation, ranging from 5 to 68 % [22]. Generally, higher lipid content could be obtained in a nitrogen-limiting or nutrient-limiting environment; however, the biomass productivity achieved in this stressed condition is usually far lower than that in normal circumstances, which results in an unchanged or even lower microalgal lipid productivity [3, 23, 24]. Since the biomass productivity of open ponds is estimated to be up to only 86.7 tons/ha/ year, much lower than what is biologically possible for microalgae [25, 26], the most important research field for photoautotrophic cultivation is the design of photobioreactors, which can leverage the incident irradiance, improve fluid dynamics, and maximize biomass production [27–30]. Merchuk et al. reviewed the ways in which integration of fluid dynamics and photosynthetic kinetics can be carried out in photobioreactors [31]. Heterotrophic Cultivation Heterotrophic cultivation is the mode in which microalgae utilize organic compounds as both energy source and carbon source. Independent of light, heterotrophic cultivation could avoid the defects associated with photolimitation in photoautotrophy; thus, higher biomass productivity can be obtained [24, 32–36]. Lipid content under heterotrophic cultivation is generally at par with or higher than that under photoautotrophic mode [34, 37], which contributes to even higher lipid productivity, thus save the cost of downstream processing. Under heterotrophic cultivation, microalgae are usually supplied with organic carbon sources and cultivated in fermenters [38]. A wide variety of organic compounds can be utilized by microalgae in heterotrophy, among which sugar/organic acid-based organic carbon sources remain the most commonly adopted and effective forms [24], although some studies revealed that cheaper sources such as corn powder hydrolysate and glycerol can also reach satisfactory yields [38, 39]. Because of the large requirement of organic compounds and bioreactors, the cost of heterotrophic cultivation is so much higher than photoautotrophy [40, 41]. Tabernero et al. comprehensively evaluated the industrial potential of biodiesel from a microalgae heterotrophic culture. A non-conservative estimation revealed no feasibility of the production plant unless biomass residues were sold; a conservative estimation showed nonviability of the process even if the residues were sold [41]. Another disadvantage related to sugar/organic acid-based heterotrophic cultivation is its vulnerability to contamination by other microorganisms, which may reduce the quality and quantity of products of interest [22]. Therefore, achieving axenic monoalgal culture is of vital importance for heterotrophic cultivation, which in turn contributes to the cost of microalgal biodiesel production. Mixotrophic Cultivation Mixotrophic cultivation is the mode in which microalgae can drive both photoautotrophy and heterotrophy and can utilize both inorganic and organic carbon sources [42, 43]. Inorganic carbon is fixed through photosynthesis which is influenced by illumination conditions, while


organic compounds are assimilated through aerobic respiration which is affected by the availability of organic carbon [44]. The main difference between mixotrophy and another scarcely observed cultivation mode, namely photoheterotrophy, which also involves light and organic carbon sources, is that photoheterotrophy requires light as the energy source, while mixotrophy can use organic compounds to achieve that. Hence, photoheterotrophic cultivation needs both organic carbon and light at the same time and is rarely used as an approach to produce microalgal biodiesel [22]. Some scientists suggested that the specific growth rate of microalgae under mixotrophic cultivation is approximately the sum of those under photoautotrophic and heterotrophic modes [45], whereas others believed that the specific growth rate in mixotrophy is not the simple combination of those in photoautotrophy and heterotrophy, and the two metabolic processes (i.e., photosynthesis for photoautotrophy and aerobic respiration for heterotrophy) affect each other under mixotrophic cultivation, which may contribute to synergistic effects and enhance biomass productivity [29, 36, 46, 47]. Since organic compounds can be utilized under mixotrophic cultivation, the growth of microalgae does not strictly depend on photosynthesis; light is not an absolute limiting factor for microalgal growth; therefore, photolimitation or photo-inhibition can be reduced in mixotrophic cultures when illumination levels are too low or too high [29, 48]. Complementing photoautotrophy with organic substrates, mixotrophic cultivation of microalgae can improve the growth rate, shorten the growth cycle, reduce biomass loss in dark hours due to pure respiration, and increase biomass productivity [48, 49]. Sometimes lipid content can be augmented as well, which leads to an even higher lipid productivity and is of great importance for microalgal biodiesel production [50]. Moreover, the CO2 released by microalgae via aerobic respiration can be trapped and reused for photosynthesis under mixotrophic cultivation, which further enhances biomass and lipid productivities [3]. Compared with heterotrophy that relies merely on organic carbon sources, mixotrophic cultivation of microalgae yields higher productivities with identical organic carbon supply. Considering the requirement of organic carbon sources contributes largely to the energy/cost input of microalgae cultivation [40], cost reduction can be achieved under mixotrophic cultivation, thus beneficial for large-scale microalgal biodiesel production.

Virtues of Mixotrophic Cultivation of Microalgae Based on the mechanisms and characteristics of the three major cultivation modes discussed above, virtues of mixotrophic cultivation are highlighted in the following section with concrete experimental results in respects of light sensitivity, biomass productivity, and lipid productivity. Other advantages of mixotrophic cultivation are also summarized. Light Sensitivity As light intensity increases, the effect of light on microalgal growth could be categorized into three phases: photolimitation in which growth rate increases with the augment of light intensity, photosaturation in which growth rate is relatively independent with light intensity, and photo-inhibition in which growth rate declines with the increase of light intensity [51]. Mixotrophically cultivated microalgae are less sensitive to light than those cultivated under photoautotrophy, no matter to which phase the intensity of illumination belongs. Within the range of photosaturation, as light intensity increased by three times, the augmentation of specific growth rate of Nostoc flagelliforme under mixotrophic cultivation


was significantly lower than that under photoautotrophic cultivation [36]. The smaller ratio of specific growth rate augmentation corresponding to light intensity increase under mixotrophic cultivation indicates that, compared with photoautotrophy, mixotrophically cultivated microalgae are less sensitive to light, and lower light intensities within photosaturation range do not markedly impede biomass productivity [43, 47]. At low light intensity that leads to photolimitation, compared with photosaturation conditions, the average specific growth rate of Synechocystis sp. PCC 6803 was observed to drop much slower in mixotrophy than in photoautotrophy [47], demonstrating that microalgae under mixotrophic cultivation are less sensitive and more tolerant toward photolimitation. Mixotrophic cultivation also displays higher tolerance toward oversaturating light intensities. Photo-inhibition of Spirulina sp. was observed at light intensities above 50 W m−2 under photoautotrophic cultivation, whereas within the scope of the experimental light intensities (0–65 W m−2), photo-inhibition of microalgae under mixotrophic cultivation was not observed [32]. Vonshak et al. [46] researched the photosynthetic responses of Spirulina platensis to extremely high light intensity (3,000 μmol photon m−2 s−1) under both photoautotrophic and mixotrophic cultivation modes. Results demonstrated that the light-dependent oxygen evolution rate and the maximum photosystem II photochemistry efficiency declined more rapidly in photoautotrophy than in mixotrophy. Reducing light intensity to normal level (50 μmol photon m−2 s−1), microalgae under mixotrophic cultivation recovered faster and to a higher extent. Being less sensitive toward various levels of light intensities, mixotrophically cultivated microalgae can better acclimate to and recover from diurnal light changes, which would alleviate the burden of artificial illumination cost. The lower light sensitivity of mixotrophic cultivation is especially advantageous for cultivating microalgae at high cell densities or with dark colored (opaque) growth medium such as wastewater, in which occasions light penetration often becomes a limiting factor [52]. Biomass Productivity With complementary organic substrates, the productivity of microalgal biomass in mixotrophy is generally much higher than that in photoautotrophy [35, 50, 53–57]. With identical organic compound supply, some strains of microalgae can achieve synergistic effect under wellcontrolled mixotrophic mode and can reach higher biomass productivities than in heterotrophic culture [36, 57]. The highest biomass productivities of Nannochloropsis oculata, Dunaliella salina, Chlorella sorokiniana, Spirulina platensis, and Scenedesmus obliquus under mixotrophic cultivation with glucose supply were 1.4 times, 2.2 times, 2.4–4.2 times, 3.8 times, and 8.7 times of that under photoautotrophic cultivation [35, 50, 54, 55]. The addition of glucose, acetate, and glycerol under mixotrophic cultivation, respectively, improved the biomass productivity of Phaeodactylum tricornutum to be 1.5-, 1.7-, and 2.5-fold of that obtained in photoautotrophy [56]. Employing glucose as organic carbon source, the average biomass productivity of mixotrophically cultivated Chlorella protothecoides was observed to be as high as 23.9 g l−1 day−1 [53], which is 1 to 2 magnitudes higher than that generally achieved in outdoor photobioreactors under photoautotrophic cultivation [22]. Bhatnagar et al. [57] investigated the biomass productivities of Chlamydomonas globosa, Chlorella minutissima, and Scenedesmus bijuga under three major cultivation modes. Experimental results indicated that with 1 % (w/v) glucose addition, biomass productivities of Chlamydomonas globosa, Chlorella minutissima, and Scenedesmus bijuga under mixotrophic cultivation were 9.4 times, 6.7 times, and 5.8 times of those under photoautotrophic cultivation and were 3.0 times, 2.0 times, and 4.4 times of those under heterotrophic


cultivation. Similar results had been achieved earlier by Yu et al. [36], who found that the highest biomass productivity of Nostoc flagelliforme was obtained through mixotrophic cultivation on glucose and was, respectively, 5.0- and 2.3-fold of that achieved in photoautotrophy and heterotrophy. Compared with other cultivation modes, the relatively high microalgal biomass productivity in mixotrophy may contribute to a higher biomass and a shorter cultivation cycle, which could make it technically easier for large-scale microalgal biodiesel production. Lipid Productivity Lipid productivity is determined by both biomass productivity and lipid content, which can be expressed as follows: lipid productivity=biomass productivity×lipid content. It is evident that, to achieve the highest possible lipid productivity, integrated effects of biomass productivity and lipid content should be taken into consideration. Since the highest levels of the above two parameters can seldom be simultaneously achieved, mixotrophy is considered to be of great advantage because much higher biomass productivity can be obtained with limited lipid content reduction. The biomass productivity and lipid productivity of several microalgal strains under mixotrophic cultivation with common organic carbon sources are listed in Table 1. Compared with photoautotrophic cultivation, the lipid productivity of mixotrophically cultivated Nannochloropsis sp. with glycerol as organic carbon source was improved by 40– 100 % [58]. Supplemented with glucose, the lipid productivities of Nannochloropsis oculata, D. salina, and Chlorella sorokiniana under mixotrophic cultivation were 1.1–1.6 times, 1.8– 2.4 times, and 4.1–8.0 times of those under photoautotrophic cultivation [32]. According to Mandal and Mallick [55], the lipid productivity of Scenedesmus obliquus under mixotrophic cultivation with 1.5 % (w/v) glucose supply could be as high as 270 mg l−1 day−1, which was approximately 50 times of that achieved in the photoautotrophic culture as control. Compared with heterotrophic cultivation, mixotrophically cultivated Chlorella protothecoides on glucose was reported to achieve 69 % higher lipid productivity [53]. Liang et al. [24] investigated the lipid production of Chlorella vulgaris under photoautotrophic, mixotrophic, and heterotrophic cultivation conditions. Experimental results indicated that with 1 % (w/v) glucose addition, the lipid productivity of Chlorella vulgaris under mixotrophic cultivation was, respectively, 1.5 times and 13.5 times of that under heterotrophic and photoautotrophic cultivation. It should be concerned that, however, not all components in total lipids can be transferred to biodiesel, since the percentage of lipid suitable for biodiesel production, namely saponifiable lipid, varies with strains and cultivating conditions; thus, the productivity of saponifiable lipid should be regarded as a more precise description of microalgal biodiesel productivity [39, 59, 60]. This, however, has not been widely analyzed. Others It has been widely observed that the chlorophyll content (dry weight of biomass) of microalgae under mixotrophic/heterotrophic cultivation decreases dramatically compared with that under photoautotrophic cultivation [35, 53, 57]. This decrease is probably due to the reduction in chlorophyll synthesis since energy can be obtained through organic compound respiration besides photosynthesis that synthetize chlorophyll. Considering the interference of chlorophyll with transesterification, an essential process in biodiesel production, such decrease improves the efficiency of downstream processing and adds value to microalgal biodiesel production. Besides, mixotrophic/heterotrophic cells generally exhibit much higher oleic acid content [33],

Appl Biochem Biotechnol Table 1 Biomass productivity and lipid productivity of several microalgal strains under mixotrophic cultivation with common organic carbon sources Microalgae strains

Organic carbon source

Biomass productivity (g l−1 day−1)

Chlamydomonas globosa Chlorella minutissima

Glucose Glucose

0.018–0.044 0.029–0.032

Chlorella protothecoidesa

Glucose

23.9 (average) 58.4 (max)

11,800 (average) [53]

Chlorella sorokiniana CCTCC M209220b

Glucose

0.58 (max)

29.0–56.0

[50]

Chlorella vulgaris #259 Glucose Dunaliella salina FACHB 435b Glucose

0.25–0.26 0.18 (max)

52.0–56.0 9.0–12.0

[24] [50]

Nannochloropsis oculata CCMP 525b

Glucose

0.20 (max)

10.0–14.0

[50]

Nannochloropsis sp.c

Glycerol 0.058–0.062

17.3–19.1

10.6–11.4

[58]

Nannochloropsis sp.c,d


16.0–17.2

6.7–7.2

[58]

Nannochloropsis sp.c,e Nannochloropsis sp.c,f

Glycerol 0.049–0.054 Glycerol 0.070–0.074

19.6–21.2 18.4–20.3

10.2–10.8 13.5–14.5

[58] [58]

Nostoc flagelliforme

Glucose

0.23–0.25

Phaeodactylum tricornutum

Glucose

0.060–0.096



Lipid content Lipid (% of dry weight) productivity (mg l−1 day−1)

Reference

[57] [57]

20.0–22.0

[36] [56] [56]


Acetate

0.069–0.11

[56]

Scenedesmus bijuga

Glucose

0.025–0.036

[57]

Scenedesmus obliquus

Glucose

0.12–0.64

Spirulina platensisb

Glucose

0.26–0.82

a

12.6–58.3

73.2–270

[55] [54]

CO2

b

Air

c

Sodium carbonate

d

Red light

e

Green light

f

Blue light

which favors the balance of oxidative stability and low-temperature properties thus promotes the quality of biodiesel. Moreover, mixotrophic cultivation is also associated with less CO2 emission than heterotrophic cultivation on the basis of per unit biomass/lipid production, since part of CO2 release can be compensated by photosynthesis. Compared with heterotrophic cultivation, mixotrophic cultivation of Chlorella protothecoides was reported to release 61.5 % less CO2 upon the same yield of lipid [53]. The reduction of CO2 emission improves the net energy balance in microalgae cultivation and may further cast positive effects on global climate change [61].

Influencing Factors of Mixotrophic Cultivation of Microalgae Both the composition and the productivity of microalgal biomass under mixotrophic cultivation are affected by various environmental and operational factors [62]. Comprehensive


consideration and successful manipulation of these factors may lead to optimum cultivating conditions and maximized productivities. Strain Selection Within the extensive boundary of microalgae, many species are mixotrophs, belonging to taxonomic groups such as cyanobacteria, chrysophyta, chlorophyta, dinophyta, diatoms, and xanthophyta [26, 36, 56, 63–65]. Selection of appropriate strains is of great importance since it intrinsically determines the quality and quantity of lipid production. High lipid productivity should be the key characteristic in selecting mixotrophic strains for microalgal biodiesel production [66]. In this regard, many microalgal strains, including some Chlorella strains, Dunaliella strains, Nannochloropsis strains, Botryococcus braunii, Pleurochrysis carterae, Scenedesmus obliquus, and Phaeodactylum tricornutum have been reported to achieve relatively high lipid productivities under mixotrophic cultivation [24, 50, 53, 55, 58, 67, 68]. Among these strains, Chlorella is a model species especially in studies on lipid accumulation [69] and is known for producing shorter-chain fatty acids (16–18 carbon length) that are suitable for transesterification in the process of biodiesel production [5, 37, 70]. To further boost lipid productivities, genetically manipulated mixotrophic strains can be selected. However, this area is currently still in its initial stage [38, 62, 71]. Carbon Sources Organic Carbon Sources Addition of organic carbon compounds could significantly enhance productivities of mixotrophic microalgae. However, the promoting effect does not always increase proportionally with augmented organic carbon dosage [48, 72]. Yields of organic compound incorporated into biomass and lipid (Ybiomass/organic compound, Ylipid/organic compound) are important parameters for biodiesel production, which generally show inverse relationships with specific organic compound uptake rates [53]. Higher yields can be achieved in organic compound limited continuous cultures [50, 73]. Various organic compounds can be utilized by microalgae under mixotrophic cultivation. Among which, glucose and acetate are the two most efficient and most frequently adopted sources. Other types of organic compound such as glycerol, fructose, sucrose, and ethanol can also enhance productivities for specific microalgal strains [35, 74]. In addition to the above pure and consequently costly organic carbon sources, utilization of much cheaper by-products, waste, and wastewater from agro-industrial sectors could also achieve satisfactory productivities for mixotrophic microalgae cultivation. Molasses, corn powder hydrolysate, crude glycerol from biodiesel industry, animal manure, agroindustrial wastewater, and municipal wastewater have been reported as organic carbon sources under mixotrophic cultivation by several microalgal strains [5, 24, 37, 44, 48, 57, 75–80]. Utilization of such cheap organic carbon sources could considerably cut down the cost of microalgae cultivation and enhance the economic feasibility of microalgal biodiesel production. Inorganic Carbon Sources Gaseous CO2 is the most commonly utilized form of inorganic carbon source for mixotrophic microalgae. Some strains are also able to utilize soluble carbonates such as Na2CO3 and


NaHCO3. Such capacity is quite advantageous in species control, since many microorganisms cannot survive in high salt content and consequently high pH environment [81, 82]. Enrichment of inorganic carbon sources has been reported to achieve higher lipid productivity [42, 83, 84]. To accomplish cost-effective results, cheap means of inorganic carbon enrichment should be adopted. This can be realized by feeding growth cultures with exhaust gases from power plants or other agro-industrial processes, in which 20–45 % of the component is CO2 [74, 85–87]. However, many strains cannot tolerate such high CO2 concentrations [17, 88]. High levels of SOX and NOX pose another question in utilizing exhaust gases, since most microalgae cannot survive under such conditions [38]. NaHCO3 from chemical CO2 fixation provides another cheap inorganic carbon source; concentration of 2–4 g l−1 is considered as sufficient for inorganic carbon enrichment [19, 89]. Illumination Although less sensitive toward light than photoautotrophy, illumination is still an important factor influencing productivities of mixotrophically cultivated microalgae. Selection of appropriate light wavelengths, light intensities, and corresponding light sources for target microalgal strains would favor higher yields. Throughout the spectrum of sunlight, only wavelengths between 400 and 700 nm (about 42.3 % of the energy from solar irradiation) can be utilized in photosynthesis by microalgae [58]. It is generally considered that wavelengths of 600–700 nm (red light) are most efficient for photosynthesis, while wavelengths of 400–500 nm (blue light) may improve the overall growth rate of mixotrophic microalgae [71, 90]. Narrowed spectrum of illumination could enhance light utilization efficiency and productivities of microalgae [91], but the cost of corresponding light sources is much higher than sunlight or the currently widely used white fluorescent lamps, which may not be applicable for large-scale microalgae cultivation [22]. Mixotrophically cultivated microalgae can acclimate to a wider range of light intensities than under photoautotrophic cultivation. Even so, excessively low or high light intensities may lead to photolimitation or photo-inhibition. Optimum light intensities should therefore match the range of photosaturation intensities [91]. Within this range, lipid especially neutral lipid tends to accumulate at elevated level with higher light intensities [92]. As cultivation process carries on, biomass concentration increases significantly. Mutual shading and photolimitation are likely to occur should light intensities be maintained at original levels [93]. To overcome this issue, measures that progressively raise illumination intensities according to biomass concentrations are recommended [54, 58, 72, 94]. Such measures could save up to 20 % of the illumination input as well [58]. High intensity intermittent light with long dark periods is also suggested as an anti-photolimitation measure. However, due to higher complexity of corresponding light sources, its application in large-scale mixotrophic microalgae cultivation is very rare [71, 95]. Because of the much higher cost of artificial illumination, it is only economical to make the most of sunlight for large-scale microalgae cultivation [35]. However, the stability, uniformity, and continuity of sunlight cannot be manipulated, which may impede the yields of mixotrophic microalgae; therefore, artificial light sources are required to complement natural illumination. Common artificial light sources include incandescent bulbs, halogen lamps, fluorescent lamps, light emitting diodes, and optical fibers [22, 71]. Nutrients According to the approximate molecular formula of microalgal biomass, i.e., CO0.48H1.83N0.11P0.01 [9], the most important influencing nutrients for microalgal biodiesel production are nitrogen and


phosphorus. For mixotrophic microalgae, nitrogen can be utilized as NO3−, NO2−, NH4+, or even as N2 [19, 96]. Nitrate, ammonium, and urea are the three most commonly adopted nitrogen sources. Among which, ammonium was observed to be the most readily taken form [97]. Urea as nitrogen source could reduce the cost of nutrient supply compared with the other two aforementioned sources. Other cheaper sources could be obtained from treated and/or untreated waste and wastewater, which contain different forms of nitrogen in large quantities [98]. Concentration of nitrogen sources has great influences on the quality and quantity of biodiesel from mixotrophic microalgae. Generally, strains under nitrogen deficiency (minimum nutrient requirements cannot be met according to formula CO0.48H1.83N0.11P0.01) display enhanced lipid content [4, 81, 92, 96]. In such case, cell proliferation is impeded because of insufficient nitrogen to synthetize enzymes and essential cell structures, but carbon is still assimilated by microalgal cells, contributing to lipid accumulation as a means of energy storage [7, 23, 68, 93]. However, because of impeded cell proliferation, biomass productivities of microalgae under nitrogen deficiency are much lower than in nitrogen sufficient environments, leading to unchanged or even lower lipid productivities [26]. To overcome such issue, a two-stage cultivating method is recommended [28, 55, 99, 100]. In the first stage, sufficient carbon and nitrogen are supplied to maximize microalgal biomass productivity, while in the second stage, sufficient carbon and deficient nitrogen are adopted to promote lipid content. Nitrogen deficiency in the second stage could also result in a change of lipid composition; the increase of triacylglycerol from free fatty acids favors microalgal biodiesel production [7, 68, 101]. Although not as influential as nitrogen, phosphorus is still an essential macro-nutrient that has significant importance to the growth and metabolism of mixotrophic microalgae [68]. It is one of the indispensible elements compromising DNA, RNA, ATP, cell membrane materials, etc., thus highly relevant to cell structures and energy transfer [81]. The most preferred forms of phosphorus for mixotrophic microalgae are inorganic phosphates such as H2PO4− and HPO42− [13]. However, affected by environmental factors such as pH, temperature, and the presence of metal ions, the precise optimum concentration of phosphorus in the growth culture is not easy to determine [73, 93, 102]. Therefore, phosphorus is usually recommended to be supplied in significant excess [9]. As it is with nitrogen, cheap phosphorus sources such as waste and wastewater can also be utilized to cut down cultivation costs. Inoculum Conditions Inoculum size and corresponding physiological stages also affect biomass and lipid productivity of mixotrophic microalgae. Although higher specific growth rates are often obtained with lower inoculum size, higher inoculum density generally displays higher biomass productivity and better acclimation to new cultures, leading to shorter lag phase and growth period [28, 103, 104]. Lipid content is generally independent of inoculum size; however, lipid composition changes with different inoculum density. It is evident that higher inoculum size often leads to increased percentage of saturated fatty acid and reduced amount of polyunsaturated fatty acid, which has positive effects on the stability and combustion of microalgal biodiesel [105]. Under the same optical density, inoculum physiological stage has significant influence on microalgal biomass productivity. Cultures inoculated with lag phase microalgae generally reach higher biomass productivities than other phases because larger number of cells with smaller sizes reproduce at higher rates [106].


Operating Modes Batch culture, fed-batch culture, and continuous culture are the three operating modes of mixotrophic cultivation. Batch culture is the most frequently adopted mode in laboratories to study the growth, metabolisms, and kinetics of mixotrophic cultivation. In this mode, all substrates (carbon sources, nutrients, etc.) are fed at the beginning of the cultivation cycle, while all the broth and products are discharged at the end of the cycle. Among the three operating modes, batch culture is the easiest and most convenient to operate; however, because of the quickly reduced substrate concentrations during cultivation time, microalgal growth rates decay fast in this mode; thus, relatively high biomass and lipid productivities cannot be achieved. To realize higher productivities, fed-batch culture is preferred and has been extensively employed in microbial industry [53]. Different to batch culture, small quantities of substrates are intermittently added in fed-batch culture, maintaining concentrations around optimum levels. Substrate restriction is greatly reduced; thus, relatively high growth rates and overall productivities throughout the cultivation cycle can be guaranteed [39]. Fed-batch culture also saves more time for system washing and inoculum preparing, which makes it more efficient than batch culture [78]. To further optimize substrate feeding under mixotrophic cultivation, continuous culture is recommended. In this mode, fresh medium (containing both substrates and microalgae) is constantly fed into the growth culture at a certain ratio, while stale medium is constantly discharged at the same ratio. Continuous operation not only minimizes the restriction of substrate but also spares the time for non-productive processes (e.g., medium feeding and discharging, microalgae inoculation, system washing, etc.) as well [78]. However, in spite of such advantages, continuous operation is rarely applied in commercial microalgal biodiesel production and requires further research, since the ratio of medium feeding is a technically intricate parameter to control [107, 108]. Outdoor and Indoor Cultivation Large-scale outdoor cultivation of microalgae has been well established, most of which carried out in open ponds [109–112], dealing with photoautotrophic microalgae for relatively high value applications [25]. For efficient biomass production to produce microalgal biodiesel, outdoor photobioreactors supplied with CO2 enriched air were studied [28, 113, 114]. Relying on organic compounds thus vulnerable to heterotrophic bacteria and other microorganisms contamination [22], mixotrophic cultivation of microalgae should be achieved in axenic cultures to obtain products of interest; thus, indoor cultivation of microalgae using enclosed photobioreactors was necessary and was extensively researched, among which tubular, flat plate, and column photobioreactors are the most popular forms [27, 29, 115]. Although higher biomass can be realized, the cost of photobioreactors is much higher than traditional raceway ponds, which handicapped the feasibility of mixotrophic microalgae cultivation for biodiesel production [116]. This part will be further discussed in later section.

Combining Mixotrophic Microalgal Biodiesel Production with Wastewater Treatment Despite its great promises for sustainable and renewable fuel production, biodiesel from microalgae under mixotrophic cultivation has rarely been applied in large-scale commercial


production due to its high costs (about 20- to 25-folds that of conventional diesel production) [40, 57, 117, 118]. Combination of wastewater treatment with microalgal biodiesel production can greatly lessen the financial pressure of mixotrophic microalgae cultivation, realizing environmental benefits as co-effects. It should be noted that, however, wastewater used for mixotrophic cultivation should be rich in biodegradable organic compounds, which will substitute sugars as organic carbon sources for microalgae [119]. Theoretical Feasibility Considerable amounts of wastewater are generated from agro-industrial sectors, most of which rich in inorganic and organic pollutants. Without proper treatment, discharge of these pollutants into the natural environment may raise a number of environmental issues, such as eutrophication, surface and/or ground water pollution, odor, and gas emissions [19]. In order to reduce the levels of pollutants (both organic and inorganic) to discharging standards, various expensive methods (biological, physicochemical, and mechanical) are required, resulting in high cost of wastewater treatment [120, 121]. To obtain satisfactory biomass and lipid productivities of microalgae under mixotrophic cultivation, large quantities of inorganic/organic carbon substrates, nutrients, and freshwater are required, all of which contribute to the high cost of microalgal biodiesel production. Moreover, the large requirement of freshwater, a precious and fast diminishing resource nowadays, may aggravate the exploiting of freshwater resources, leading to worsened situation of global freshwater insufficiency [57]. Combination of mixotrophic microalgal biodiesel production with wastewater treatment serves as an effective way to achieve reduced input for both wastewater treatment and microalgae cultivation, since wastewater readily contains available water source and major substrates required by microalgae proliferation [52, 67, 122]. Craggs et al. [123] estimated the required power of wastewater treatment combined with mixotrophic microalgae cultivation to be 50–110 kWh/ML of wastewater, much lower than the 230–960 kWh/ML that required in conventional activated sludge treatment. Mulbry et al. [76] also concluded the annual operation costs of combining dairy manure treatment with microalgae cultivation to be well below the costs for upgrading existing wastewater treatment plants in sensitive watersheds. Supplementing wastewater with exhaust gas may further promote microalgal biomass accumulation, realizing exhaust gas mitigation at the same time [102]. An ideal combination of wastewater treatment with microalgal biodiesel production could be realized with microalgae cultivating system located near a waste/wastewater treatment plant, shortening the route and cost for wastewater/gas transportation (displayed in Fig. 2). Relationship between mixotrophic microalgae and heterotrophic bacteria in wastewater should be noted, since bacteria exist in wastewater and sterilizing wastewater is not economical for largescale microalgal biodiesel production. Studies have revealed that the algal–bacterial consortium in wastewater is more efficient in both microalgal biomass growth and pollutants removal, since bacteria has a wider range of digestible organics and can transform organic compounds into forms microalgae are capable of utilizing [117, 124–126]. This consortium, however, may complicate the downstream processes for crude biodiesel, since most bacteria produce complex lipoid and few of them can produce lipid available as feedstock for biodiesel production [127]. Current Development Research on microalgae cultivation as wastewater treatment processes started as early as 1950s [128]. Although the initial purpose of introducing microalgae to wastewater treatment process


Fig. 2 Conceptual process of combining mixotrophic microalgal biodiesel production with wastewater treatment

was to realize tertiary treatment focusing on nutrients removal [52], it was further observed that microalgae could also remove organic pollutants from sewage efficiently [129]. Combination of mixotrophic microalgal biodiesel production with wastewater treatment has been tested on various wastewater streams, including concentrated and unconcentrated municipal wastewater, digested and/or undigested animal manure, and agricultural runoff. Microalgae from the Chlorella family display excellent adaptation in wastewater and can achieve high biomass productivity, thus are the most commonly used strains for simultaneous biodiesel production and wastewater treatment [80]. Recent studies on mixotrophic microalgae cultivation with wastewater for biodiesel production are summarized in Table 2. Bottlenecks and Difficulties Promising and extensively researched as it is, commercially viable systems producing microalgal biodiesel using wastewater is currently limited. One major issue is the mismatch of geographic locations of biodiesel refineries and wastewater treatment plants; infrastructures such as effluent storage tanks and transport systems should be further addressed to facilitate the utilization of wastewater as microalgae cultivation medium [130]. Also, wide variations in biomass composition (i.e., lipid content and fatty acid composition) exist in wastewater cultivated mixotrophic microalgae, caused by the heterogeneity among different types or even the same type of wastewater. This would be a challenge for producing microalgal biodiesel to comply with existing standards [8], since important parameters of biodiesel such as oxidation

Digested dairy manure

Hydrolysates from acid hydrolysis of oil crop biomass residues

Acidogenically digested swine wastewater

Post-hydrothermal liquefaction wastewater of Spirulina, diluted by municipal wastewater

Municipal wastewater

Effluent mixes from municipal septic and secondary sedimentation tank

Chlorella sp.

Chlorella sp.

Chlorella sp.

Chlorella spp., Scenedesmus obliquus, and cyanobacteria

Chlorella vulgaris

Chlorella vulgaris

Industrial wastewater (treated and untreated)

Poultry litter extract

Hydrolysates from acid hydrolysis of oil crop biomass residues

Scenedesmus bijuga

Scenedesmus bijuga

Scenedesmus sp.

0.54 (max)

0.049–0.051

0.054–0.070

0.033–0.037

50–59

11.8–12.0

12.2–15.2

0.028–0.038

Carpet and rug wastewater with CO2 enrichment


Dunaliella tertiolecta

Pleurochrysis carterae

13.0–32.0

18.0 (average)

14.7–20.1

25.0–27.2

31–38

9.0–13.7

17.0–18.1

0.050–0.103

0.005–0.015

0.020–0.035

0.111

0.60 (max)

0.073–0.090

0.016–0.023

12.9–19.6

0.153–0.382

Highly concentrated municipal wastewater

5.6–24.1

14.4–24.2

0.035–0.163


Concentrated municipal wastewater stream with/without CO2 enriched aeration

Chlorella protothecoides

0.042–0.046

Chlorella protothecoide


Chlorella minutissima

0.030–0.046

Chlorella saccharophila

Highly concentrated municipal wastewater


Chlorella kessleri

Chlorella minutissima

4.0–4.4

4.3–4.6

13.4–16.0

1.0 (average)

2.9–7.0

27.8–30.2

8.1–10.0

2.7–4.2

19.8–74.4

2.6–39.2

29.6–91.0

[137]

[57]

[57]

[67]

[67]

[139]

[138]

[117]

[122]

[137]

[103]

[67]

[52]

[80]

[57]

[57]

[52]

0.206–0.400

[57] [80]

4.5–57.4

[67] [136]

0.043–0.222

8.0–25.9

3.5–4.5 16.8

[44]

Reference

Concentrated municipal wastewater stream with/without CO2 enrichment

Chlorella kessleri

19.8

16.5–46.9

Lipid productivity (mg l−1day−1)

[57]

0.001–0.024

0.085 (average)

9.5–13.2

18.7–20.8

0.083–0.226 0.034–0.037

Lipid content (% of dry weight)

Biomass productivity (g l−1 day−1)

0.031–0.035


Livestock wastewater

Botryococcus braunii



Botryococcus braunii

Chlamydomonas globosa

Concentrated municipal wastewater stream with/without CO2 enrichment

Auxenochlorella protothecoides

Chlamydomonas globosa

Cultivation medium

Microalgae species

Table 2 Productivities of several microalgal strains under mixotrophic cultivation with wastewater



stability and cold-flow properties are closely related to fatty acid composition and their degree of saturation [11]. Although cultivating cost can be largely reduced by utilizing wastewater, production cost of microalgal biodiesel still needs to be lowered by nearly an order of magnitude to meet the requirement of current biodiesel market [131]. The major cost concerning microalgal biodiesel production and wastewater treatment is related to biomass harvesting. Current mainstream harvesting process by means of centrifugation contributes to about 30 % of the total cost and is apparently not cost-effective [11, 124]. Another bottleneck that hinders the large-scale application of microalgal biodiesel production-based wastewater treatment process is its relatively long hydraulic retention time compared with conventional activated sludge process [129]. Other shortcomings include the existence of some hazardous compounds that may impede the growth of microalgae and challenge of producing value-added co-products to offset the high cost [81, 93].

Strategies for Future Commercial Production High Performance Strain Screening and Engineering Ideal strains for mixotrophic cultivation for biodiesel production using wastewater should be able to obtain high biomass densities and high lipid content, have fatty acid composition suitable for biodiesel production, have wide range of available carbon sources and limited nutrient requirements, and to be tolerant toward wastewater composition variation as well as other environmental changes such as light intensity, temperature, pH values, and shear stresses. The abilities of dominating among wild strains in outdoor large-scale cultivation systems, displaying self-flocculation and other specialties that may simplify biomass harvesting procedures, and producing value added co-products are also necessary for ideal mixotrophic strains [11, 65, 131]. To realize the above requirements as far as possible, screening and engineering of appropriate microalgal strains should be extensively developed. Some local strains isolated from extreme habitats could achieve significant improvement in lipid content, lipid composition, self-dominance, and self-flocculation [19]. With the aid of recently developed efficient screening techniques such as flow cytometry, time of screening could be largely reduced [7]. Application of genetic engineering in microalgal biodiesel production can intrinsically improve the performance of microalgae. By promoting the expression of acetyl-coA carboxylase gene, capacity of lipid accumulation in microalgae can be significantly improved [4, 132]; by reducing the size of chlorophyll antenna of microalgal cells, photosynthetic efficiency could be enhanced, and photo-inhibition could be avoided within a wider range of light intensities [71]. Moreover, application of genetic engineering could enable the production of value added co-products such as proteins and metabolites [38, 133], leading to enhanced economic feasibility of microalgal biodiesel production. However, stability of desirable trait through many generations and possibility of unintended horizontal gene transfer to other organisms should be concerned [8]. Combination of CO2 Mitigation Currently, more than 30×109 tons of CO2 is released from fossil fuel combustion, only 12× 109 tons of which can be removed through natural processes [85]. Utilization of such exhaust


gas for microalgae cultivation would not only improve microalgal productivity but would also contribute to CO2 mitigation and worldwide sustainable development. Being capable of producing 280 tons of dry biomass/ha/year while realizing roughly 513 tons of CO2 fixation at the same time, microalgae are perfect candidates for CO2 mitigation [87]. Throughout the process of mixotrophic microalgal biodiesel production and wastewater treatment, CO2 mitigation can be realized in two major ways: (a) producing biodiesel to replace the fossil fuels currently used and (b) assimilating organic pollutants into cellular components such as lipid and carbohydrate rather than degrading them into CO2 (as conventional activated sludge process does) [129]. Besides, microalgae could also enhance the quality of biogas produced in biomass digestion procedure by reducing its CO2 content [131]. Promising as it seems, combination of CO2 mitigation with mixotrophic microalgal biodiesel production and wastewater treatment requires necessary design of the mass transfer unit (and photo bioreactor design in general) and relatively tolerant strains [134]. Otherwise, exhaust gases from power plants or other agro-industrial sectors should be pretreated before aerated into growth cultures, since excessive levels of CO2, SOX, NOX, and temperature may generate negative effects on the growth of mixotrophic microalgae [74]. Cost-Effective Photobioreactor Development and Scale-Up Selection of photobioreactors has great influences on illumination and mass transfer conditions, biomass, and lipid productivities and may considerably affect the cost of mixotrophic microalgae cultivation and downstream processing. Photobioreactors can be designed as open or enclosed systems. Open systems are mostly operated under solar illumination, characterized by moderate surface to volume ratios (3–10 m−1), represented by stabilization ponds and high rate algal ponds [93]. One of the most outstanding advantages of open systems is their technical and economical convenience to scale-up and to operate. However, light utilization and mass transfer is poor in open systems, resulting in low biomass and lipid productivities. Other limitations of open systems include large land requirements, evaporative losses, diffusion of gases (mostly CO2, sometimes NH3) into the atmosphere, and high risks of contamination by other microorganisms [43, 135]. Enclosed photobioreactors are characterized by large surface to volume ratios (25–125 m−1). There are various types of enclosed photobioreactors, generally categorized into tubular, flat plate, and column photobioreactors [38]. Although available for better control of cultivation conditions, higher productivities and efficiencies, and less risk of contamination, enclosed photobioreactors are generally more costly to construct and operate [93] and are technically very difficult to scale up, mainly due to the challenges in maintaining optimum cultivation conditions in expanded photobioreactors. Moreover, most enclosed photobioreactors are complemented with artificial light sources; this would result in high illumination costs and may additionally offset the relief on energy crisis, since fossil fuels are generally required for power generation [135]. Future development should focus on innovative designs of photobioreactors that are technically and economically feasible to scale-up. This may be realized through utilizing costeffective illuminating techniques and renewable sources such as solar energy, wind energy, wastewater, exhaust gas, biogas, and waste heat. A real-time smart on-line monitoring system is also necessary for continuous large-scale microalgae production in photobioreactors [101].


Valuable Co-product Production Apart from biodiesel, a microalgae biorefinery can also produce biohydrogen, biogas, bioethanol, etc. Even the microalgal biomass residue that remains after lipid extraction can be potentially utilized to produce methane [8]. In addition to producing renewable energy, microalgae are also promising feedstocks in food, feed, pharmaceutical, and cosmetic industries for producing polysaccharides, proteins and pigments, etc. [101]. Such valuable coproducts would offset the currently high cost of microalgal biodiesel production and would improve its economic feasibility. However, large disparities of market requirements of these products should be taken into consideration, to make the concept of such algal biorefineries plausible.

Conclusions Mixotrophic cultivation of microalgae for biodiesel production provides a very promising alternative for producing renewable biofuels in order to overcome the increasingly fierce energy crisis. The mixotrophic mode of cultivation is advantageous than other cultivation conditions mainly in terms of low light sensitivity, high biomass productivity, and high lipid productivity. Other virtues include low organic substrate consumption, favorable biomass composition for biodiesel conversion, and low CO2 emission. Various influencing factors should be comprehensively manipulated so as to optimize the biomass and lipid productivities of mixotrophic microalgae. Combination of mixotrophic microalgal biodiesel production with wastewater treatment can greatly lessen the financial pressure of microalgae cultivation, realizing environmental benefits as co-effects. Current development and bottlenecks of this combination are summarized; future strategies such as high performance strain selection, combination of CO2 mitigation, cost-effective photobioreactor development and scale-up, and valuable co-product production could further enhance the viability of commercial microalgal biodiesel production in a technically available, economically feasible, and environmentally sustainable way. Acknowledgments Financial supports from Chinese National Key Technology R&D Program during the Eleventh Five-Year Plan Period (2009BAC57B01) are gratefully acknowledged.

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Salinity stress induced lipid synthesis to harness biodiesel during dual mode cultivation of mixotrophic microalgae.

Application of agar liquid-gel transition in cultivation and harvesting of microalgae for biodiesel production.

Heterotrophic microalgae cultivation to synergize biodiesel production with waste remediation: progress and perspectives.

Mixotrophic cultivation of oleaginous Chlorella sp. KR-1 mediated by actual coal-fired flue gas for biodiesel production.

Isolation and characterization of microalgae for biodiesel production from seawater.

Enzymatic reactors for biodiesel synthesis: Present status and future prospects.

Biodiesel production from indigenous microalgae grown in wastewater.

Development and validation of a screening procedure of microalgae for biodiesel production: application to the genus of marine microalgae Nannochloropsis.

Isolation of novel microalgae from acid mine drainage and its potential application for biodiesel production.

Isolation and characterization of microalgae for biodiesel production from Nisargruna biogas plant effluent.

Concurrent extraction and reaction for the production of biodiesel from wet microalgae.

Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load.

Simultaneous treatment of food-waste recycling wastewater and cultivation of Tetraselmis suecica for biodiesel production.

Selection of microalgae for biodiesel production in a scalable outdoor photobioreactor in north China.

Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2).

Functionalized Fe3O4@silica core-shell nanoparticles as microalgae harvester and catalyst for biodiesel production.

Screening microalgae native to Quebec for wastewater treatment and biodiesel production.

Al2O3 as heterogeneous catalyst for biodiesel production via in situ transesterification from microalgae.

Cultivation of Desmodesmus subspicatus in a tubular photobioreactor for bioremediation and microalgae oil production.

Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production.

Strategies for Lipid Production Improvement in Microalgae as a Biodiesel Feedstock.

Enhancing growth and lipid production of marine microalgae for biodiesel production via the use of different LED wavelengths.

Microalgae-mediated simultaneous treatment of toxic thiocyanate and production of biodiesel.

Cultivation of Isochrysis galbana in phototrophic, heterotrophic, and mixotrophic conditions.