**2. Biodiesel from microalgae**

Spoehr and Milner in 1949 demonstrated that the lipid content increased in the green microalga *Chlorella* under nitrogen starvation [1]. However, it was not until the early 1970s, with the oil embargo and the increase in energy prices, that the concept of lipids from microalgae for biofuel production gained attention [2]. Despite decades of research, biodiesel from microalgae has yet to be introduced to the gas station near you. It has been reported that microalgae can produce around 20–50% of their dry weight as lipids [3, 4], with only one research article suggesting as high as 80% [5]. The amount of lipid produced by a variety of species under optimum growth conditions is summarized in **Table 1**. The ability to synthesize a higher percentage of their dry weight as lipids makes microalgae the highest producer of


**213**

65 g m<sup>−</sup><sup>2</sup> ∙ day<sup>−</sup><sup>1</sup>

year<sup>−</sup><sup>1</sup>

never exceeded 100 tons ha<sup>−</sup><sup>1</sup>

amount of 224 tons ha<sup>−</sup><sup>1</sup>

280 tons ha<sup>−</sup><sup>1</sup>

**Table 1.**

*Physiological Limitations and Solutions to Various Applications of Microalgae*

**Microalga Taxa %Lipid %Protein %CHO Growth** 

*Nannochloropsis* Chlorophyceae 46–68 1.05 [6] *Nannochloropsis granulata* Chlorophyceae 24–28 18–34 27–36 [10]

*Nannochloropsis salina* Chlorophyceae 59.8 24.3 15.9 1.05 [6] *Nannochloropsis* sp. Chlorophyceae 64 1.04 [6] *Navicula acceptala* Bacillariophyceae 32–48 3.8 [6] *Nitzschia* sp. Bacillariophyceae 27 36 16 [6] *Nitzschia dissipata* Bacillariophyceae 66 12.6 9.3 1.32 [6] *Nitzschia closterium* Bacillariophyceae 13 26 10 [8] *Oocystis pusilla* Chlorophyceae 10.5 39 37 [6] *Phaeodactylum tricornutum* Bacillariophyceae 16–50 31–35 11–17 1.96 [6, 8,

*Porphyridium cruentum* Rhodophyceae 9–14 28–39 40–57 [7] *Prymnesium parvum* Prymnesiophyceae 22–38 28–45 25–33 [7] *Scenedesmus obliquus* Chlorophyceae 12–14 50–56 10–17 [7] *Scenedesmus quadricauda* Chlorophyceae 1.9 47 [7] *Scenedesmus dimorphus* Chlorophyceae 16–40 8–18 21–52 [7] *Skeletonema costatum* Bacillariophyceae 10 25 5 [8] *Spirogyra* sp. Chlorophyceae 11–21 6–20 33–64 [7] *Spirulina maxima* Cyanophyceae 6–7 60–71 13–16 [7] *Spirulina platensis* Cyanophyceae 4–9 46–63 8–14 [7] *Synechococcus* sp. Cyanophyceae 11 63 15 [7] *Tetraselmis sp.* Chlorophyceae 18 46 36 2.1 [6] *Tetraselmis chuii* Chlorophyceae 20 2.1 [6] *Tetraselmis maculate* Chlorophyceae 3 52 15 [7] *Tetraselmis succia* Chlorophyceae 15–33 2.1 [6] *Thalassiosira pseudonana* Bacillariophyceae 19 34 9 [8]

39.9

lipid per unit mass and arguably the most practical choice for the production of biodiesel. Theoretical estimates of maximum biomass production vary from 24 to

*Summary of macromolecular composition and growth rate of microalgae belonging to various taxa.*

estimates of biomass and 80% of the biomass composition as lipid, the maximum

However, the lipid produced has to be transesterified to fatty acid methyl esters, which then can be used as biodiesel, which can result in some loss. However, earlier studies have reported up to 96% recovery of lipids through direct transesterification

biodiesel can be produced. It is important to note that a recent study on *Scenedesmus accuminatus* using open and polyhouse raceway ponds estimated around 2.1 tons

year<sup>−</sup><sup>1</sup>

[12]. With 4% loss by transesterification, a maximum of 215 tons ha<sup>−</sup><sup>1</sup>

year<sup>−</sup><sup>1</sup>

[7]. Benedetti et al. have reported a theoretical maximum up to

of biomass but also noted that the actual cultivation record

[11]. Even assuming the theoretical maximum

year<sup>−</sup><sup>1</sup>

**rate d<sup>−</sup><sup>1</sup>**

**Source**

[6]

10]

of

of lipids is what can theoretically be produced.

*DOI: http://dx.doi.org/10.5772/intechopen.90206*

*Nannochloropsis oculata* Chlorophyceae 17.8–


#### *Physiological Limitations and Solutions to Various Applications of Microalgae DOI: http://dx.doi.org/10.5772/intechopen.90206*

#### **Table 1.**

*Microalgae - From Physiology to Application*

**2. Biodiesel from microalgae**

*Amphiprora hyaline* (ENTOM3)

of microalgal products. This includes existing sources (fossil fuels), technological development, and physiological limitation. In this chapter, we will describe the various known physiological limitations associated with productions of various

Spoehr and Milner in 1949 demonstrated that the lipid content increased in the green microalga *Chlorella* under nitrogen starvation [1]. However, it was not until the early 1970s, with the oil embargo and the increase in energy prices, that the concept of lipids from microalgae for biofuel production gained attention [2]. Despite decades of research, biodiesel from microalgae has yet to be introduced to the gas station near you. It has been reported that microalgae can produce around 20–50% of their dry weight as lipids [3, 4], with only one research article suggesting as high as 80% [5]. The amount of lipid produced by a variety of species under optimum growth conditions is summarized in **Table 1**. The ability to synthesize a higher percentage of their dry weight as lipids makes microalgae the highest producer of

**Microalga Taxa %Lipid %Protein %CHO Growth** 

*Amphora sp.* Bacillariophyceae 13.6 17.3 74.9 5.1 [6]

*Anabaena cylindrical* Cyanophyceae 4–7 43–56 25–30 [7] *Ankistrodesmus falcatus* Chlorophyceae 40.3 14.3 18.3 2.89 [6] *Boekelovia* sp. Chrysophyceae 20.7 3.43 [6] *Botryococcus braunii* Chlorophyceae 54.2 20.6 14.3 1.8 [6] *Chaetoceros* sp. Bacillariophyceae 22.2 31.9 43 4.3 [6] *Chaetoceros calcitrans* Bacillariophyceae 16 34 6 [8] *Chaetoceros gracilis* Bacillariophyceae 7 12 5 [8] *Chaetoceros muelleri* Bacillariophyceae 31 59 10 [9] *Chlamydomonas reinhardtii* Chlorophyceae 21 48 17 [7] *Chlorella* sp. Chlorophyceae 34–48 19–31 1.33 [6] *Chlorella ellipsoidea* Chlorophyceae 8.9 26.1 26.3 5.3 [6] *Chlorella protothecoides* Chlorophyceae 15–56 [6] *Chlorella pyrenoidosa* Chlorophyceae 2 57 26 [7] *Chlorella vulgaris* Chlorophyceae 14–22 51–58 12–17 [7] *Cyclotella* sp. Bacillariophyceae 42.1 16.4 10.2 5.1 [6] *Dunaliella bioculata* Chlorophyceae 8 49 4 [7] *Dunaliella salina* Chlorophyceae 6 57 32 [7] *Dunaliella tertiolecta* Chlorophyceae 15–43 2.58 [6] *Euglena gracilis* Chlorophyceae 14–20 39–61 14–18 [7] *Isochrysis aff. Galbana* Prymnesiophyceae 26 23.3 20.5 2.83 [6]

24.4

*Nannochloris* Chlorophyceae 31–63 3.19 [6]

Bacillariophyceae 22–37 2.3 [6]

**rate d<sup>−</sup><sup>1</sup>**

25.5 3.1 [6]

**Source**

microalgal products in a commercial scale and list possible solutions.

**212**

*Monoraphidium* sp. Chlorophyceae 17.9–

*Summary of macromolecular composition and growth rate of microalgae belonging to various taxa.*

lipid per unit mass and arguably the most practical choice for the production of biodiesel. Theoretical estimates of maximum biomass production vary from 24 to 65 g m<sup>−</sup><sup>2</sup> ∙ day<sup>−</sup><sup>1</sup> [7]. Benedetti et al. have reported a theoretical maximum up to 280 tons ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> of biomass but also noted that the actual cultivation record never exceeded 100 tons ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> [11]. Even assuming the theoretical maximum estimates of biomass and 80% of the biomass composition as lipid, the maximum amount of 224 tons ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> of lipids is what can theoretically be produced. However, the lipid produced has to be transesterified to fatty acid methyl esters, which then can be used as biodiesel, which can result in some loss. However, earlier studies have reported up to 96% recovery of lipids through direct transesterification [12]. With 4% loss by transesterification, a maximum of 215 tons ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> of biodiesel can be produced. It is important to note that a recent study on *Scenedesmus accuminatus* using open and polyhouse raceway ponds estimated around 2.1 tons

ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> , which is 100 times lower than the theoretical maximum [13]. Another study with *Chlorella* sp. L1 and *Monoraphidium dybowskii* Y2 using batch and semicontinuous mode in a raceway pond resulted in lipid productivities of 13.91 and 14.45 ton ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> in semicontinuous mode [14], tenfold lower than the theoretical estimates. This discrepancy between theoretical estimates and laboratory and field tests are primarily due to physiological limitations, especially carbon fixation and light absorption [15, 16].

The current demand of fossil fuel is approximately 100 million barrels per day (~11,563 million liters per day), which is only expected to increase with time (105 million barrels per day by 2021) [17]. To meet this magnitude of global demand, assuming 15 ton ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> lipid productivity from microalgae, around 276 million hectares of land would be required for microalgal cultivation every day. This huge demand can be reduced by increasing the lipid productivity of microalgae by minimizing energy and carbon wasting physiological processes in the cell and redirecting it towards lipid synthesis. RUBISCO, the enzyme catalyzing the dark reaction of photosynthesis, wherein the atmospheric or dissolved CO2 is converted into organic triose phosphate, has a lower affinity to CO2. In addition, oxygen is a competitive substrate for RUBISCO, catalyzing the reaction of photorespiration wherein energy and NADPH are utilized and fixed CO2 is released. Therefore, during CO2 limitation, some species of microalgae utilize a carbon concentration mechanism (CCM) that increases the concentration of CO2 at the site of RUBISCO, leading to the catalysis of the dark reaction of photosynthesis. Although the CCM is an ingenious mechanism evolved to address the lower CO2 affinity of RUBISCO and the competition by oxygen, the process consumes one to two molecules of ATP [18]. This energy can instead be redirected towards lipid synthesis by simply bubbling CO2 into the cultures [19] or by selection CCM lacking microalgae species.

Finkel et al. found that the median macromolecular composition of nutrientsufficient exponentially growing microalgae is 17.3% lipid, 32.2% protein, and 15% carbohydrates [20]. For biofuels, lipids are the most important cellular fraction. *Botryococcus braunii* has the highest lipid as percent dry weight, 43%, while *Tetraselmis suecica* and *Dunaliella tertiolecta* have the least (<10%). Also in speciesspecific differences (**Table 1**), Finkel et al. showed that there are some phylogenetic differences [20]. Cyanobacteria have the lowest lipid content (11.7%; dry wt), while the Bacillariophyta (diatoms) had the highest (18.6–21.3%; dry wt). In fact, in most cases, these median values are all significantly lower than those values reported in **Table 1**, which are up to 60% (w/w) for lipid, protein, and carbohydrate. The values at the higher end of the spectrum are typically induced after some kind of stress (e.g., light, nutrient) is placed on the microalgae. Nutrient stress is most often used to induce lipid production, particularly nitrogen [5, 21, 22]. These large lipid stores are thought to provide a growth advantage under variable resource supplies [23].

Light harvesting systems of microalgae are usually capable of absorbing between 350 and 700 nm; however much of the radiation extending out to 1100 nm remains unused. In addition, Raven et al. calculated a 22% loss of energy even within the spectrum of 350–700 nm [24]. However, anoxygenic photosynthetic bacteria possess bacteriochlorophylls capable of absorbing light up to 1050 nm and perform photochemistry [19]. These bacteriochlorophyll-based photosystem can be engineered into microalgae. By replacing photosystem I, it would extend the range of absorbed photons that can be used for photochemistry [25], therefore the energy available for lipid synthesis. It has been proposed that smaller cells can photosynthetically perform better larger cells [26, 27], primarily attributing to the "package effect." Package effect refers to the inverse relationship between light harvesting ability of the cells and the cell volume. However, studies have shown that larger cells tend to decrease the antennae size, thereby counteracting the negative impacts of

**215**

(83,700,000 μm3

and 28–200 μm3

*Physiological Limitations and Solutions to Various Applications of Microalgae*

package effect [28, 29]. Moreover, several studies have shown that decreasing the antennae size significantly increased the photosynthetic efficiencies [30, 31]. This could be due to decreased photoinhibition and decreased allocation of resources towards light harvesting systems [19]. However, the hypothesis that larger cells would be less photosynthetically efficient than smaller cells due to package effect was recently disproved by a recent study by Malerba et al. [32], which showed that larger cells developed compensatory mechanisms by reducing the antenna size, increasing the connectivity between the photosynthetic units, and decreasing the levels of photo-protective pigment β-carotene. This in turn minimized the negative significances of larger cell volume-induced package effects. Exposure to extreme conditions such as photoinhibitory light levels, UVA and UVB radiation, and nutrient and temperature stress could result in oxidative damage to cellular components especially proteins. These kinds of damage result in breakdown of the damage component and re-synthesis of these components or adaptive changes to the cellular components. These changes however have a cost in terms of energy (i.e., ATP, NADPH) and resources which could have been directed towards biomass and lipid synthesis. Protein turnover during scotophase has been shown to consume about one-third of the total respiratory ATP production [33]. Exposure to nitrogen-limiting conditions could result in upregulation of nitrogenase in diazotrophic cyanobacteria, which in turn could result in 10% decrease in growth rates [34, 35]. These are avoidable energy and resource expenditure, which can be easily prevented by providing optimal light, nutrient, and temperature during mass cultivation.

Lipids are not secreted/excreted actively by all microalgae, with exception of *Botryococcus* sp. [36]. This constrains the maximum amount of lipid that is synthesized and stored in a microalgal cells, as the largest reported cell volume for a microalgae is for species *Noctiluca scintillans* belonging to the class Dinophyceae

ogy belong to the class Chlorophyceae such as *Chlorella* sp., *Haematococcus* sp., and

[38], and the species belonging to class Bacillariophyceae such as *Phaeodactylum tricornutum* and *Thalassiosira pseudonana* which have cell volumes of ~60–100 μm3

inverse relationship with the volume of a cell, by a factor of 3/4 [38]. This further constrains the maximum amount of lipids that can be synthesized in a microalgal mass culture. However, if the lipids were secreted in a manner similar to EPS, physiological limitations such as cell volume or maximum density would not play a constraining role on the maximum amount of lipid that can be synthesized in an algal mass culture. Microalgal species like *Botryococcus* sp. has already been shown to actively secrete lipids that can be converted into fuel-grade biodiesel. However, their slower growth rates limit the maximum lipids that can be synthesized. Microalgal species like *Scenedesmus* has been shown to grow as fast as 1.53 per day [40]. Therefore, genetic engineering techniques to integrate the lipid-secreting trait

Phototrophic modes of cultivation depend on two critical factors, namely, light and carbon dioxide [16]. Therefore, microalgae grown phototrophically can either be limited in light or carbon dioxide or both, limiting the maximum cellular density one can achieve using this mode of cultivation. In addition, various inefficiencies described above with light-harvesting abilities of microalgae, lower affinity for CO2 of RUBISCO, cost associated with protein turnover, and photoinhibition can be simply avoided by growing microalgae heterotrophically and mixotrophically. Extensive amount of research suggests enhanced biomass production under heterotrophic and mixotrophic modes of cultivation, increasing the cellular density to as much as 4 to 5 times [41, 42]. Further modification in these modes of cultivation such as using

*Scenedesmus* sp., which have cell volumes of 34.5, 48, and 26,200 μm3

into the fast-growing *Scenedesmus* sp. could be a possible solution.

) [37]. The most commonly used microalgal species in biotechnol-

[37, 39]. On the other hand, the maximum cellular density has an

, respectively

*DOI: http://dx.doi.org/10.5772/intechopen.90206*

#### *Physiological Limitations and Solutions to Various Applications of Microalgae DOI: http://dx.doi.org/10.5772/intechopen.90206*

*Microalgae - From Physiology to Application*

year<sup>−</sup><sup>1</sup>

year<sup>−</sup><sup>1</sup>

and light absorption [15, 16].

assuming 15 ton ha<sup>−</sup><sup>1</sup>

, which is 100 times lower than the theoretical maximum [13]. Another

in semicontinuous mode [14], tenfold lower than the theoreti-

lipid productivity from microalgae, around 276 mil-

study with *Chlorella* sp. L1 and *Monoraphidium dybowskii* Y2 using batch and

semicontinuous mode in a raceway pond resulted in lipid productivities of 13.91 and

The current demand of fossil fuel is approximately 100 million barrels per day (~11,563 million liters per day), which is only expected to increase with time (105 million barrels per day by 2021) [17]. To meet this magnitude of global demand,

cal estimates. This discrepancy between theoretical estimates and laboratory and field tests are primarily due to physiological limitations, especially carbon fixation

lion hectares of land would be required for microalgal cultivation every day. This huge demand can be reduced by increasing the lipid productivity of microalgae by minimizing energy and carbon wasting physiological processes in the cell and redirecting it towards lipid synthesis. RUBISCO, the enzyme catalyzing the dark reaction of photosynthesis, wherein the atmospheric or dissolved CO2 is converted into organic triose phosphate, has a lower affinity to CO2. In addition, oxygen is a competitive substrate for RUBISCO, catalyzing the reaction of photorespiration wherein energy and NADPH are utilized and fixed CO2 is released. Therefore, during CO2 limitation, some species of microalgae utilize a carbon concentration mechanism (CCM) that increases the concentration of CO2 at the site of RUBISCO, leading to the catalysis of the dark reaction of photosynthesis. Although the CCM is an ingenious mechanism evolved to address the lower CO2 affinity of RUBISCO and the competition by oxygen, the process consumes one to two molecules of ATP [18]. This energy can instead be redirected towards lipid synthesis by simply bubbling CO2 into the cultures [19] or by selection CCM lacking microalgae species.

Finkel et al. found that the median macromolecular composition of nutrientsufficient exponentially growing microalgae is 17.3% lipid, 32.2% protein, and 15% carbohydrates [20]. For biofuels, lipids are the most important cellular fraction. *Botryococcus braunii* has the highest lipid as percent dry weight, 43%, while *Tetraselmis suecica* and *Dunaliella tertiolecta* have the least (<10%). Also in speciesspecific differences (**Table 1**), Finkel et al. showed that there are some phylogenetic differences [20]. Cyanobacteria have the lowest lipid content (11.7%; dry wt), while the Bacillariophyta (diatoms) had the highest (18.6–21.3%; dry wt). In fact, in most cases, these median values are all significantly lower than those values reported in **Table 1**, which are up to 60% (w/w) for lipid, protein, and carbohydrate. The values at the higher end of the spectrum are typically induced after some kind of stress (e.g., light, nutrient) is placed on the microalgae. Nutrient stress is most often used to induce lipid production, particularly nitrogen [5, 21, 22]. These large lipid stores are thought to provide a growth advantage under variable resource supplies [23]. Light harvesting systems of microalgae are usually capable of absorbing between 350 and 700 nm; however much of the radiation extending out to 1100 nm remains unused. In addition, Raven et al. calculated a 22% loss of energy even within the spectrum of 350–700 nm [24]. However, anoxygenic photosynthetic bacteria possess bacteriochlorophylls capable of absorbing light up to 1050 nm and perform photochemistry [19]. These bacteriochlorophyll-based photosystem can be engineered into microalgae. By replacing photosystem I, it would extend the range of absorbed photons that can be used for photochemistry [25], therefore the energy available for lipid synthesis. It has been proposed that smaller cells can photosynthetically perform better larger cells [26, 27], primarily attributing to the "package effect." Package effect refers to the inverse relationship between light harvesting ability of the cells and the cell volume. However, studies have shown that larger cells tend to decrease the antennae size, thereby counteracting the negative impacts of

ha<sup>−</sup><sup>1</sup>

year<sup>−</sup><sup>1</sup>

14.45 ton ha<sup>−</sup><sup>1</sup>

**214**

package effect [28, 29]. Moreover, several studies have shown that decreasing the antennae size significantly increased the photosynthetic efficiencies [30, 31]. This could be due to decreased photoinhibition and decreased allocation of resources towards light harvesting systems [19]. However, the hypothesis that larger cells would be less photosynthetically efficient than smaller cells due to package effect was recently disproved by a recent study by Malerba et al. [32], which showed that larger cells developed compensatory mechanisms by reducing the antenna size, increasing the connectivity between the photosynthetic units, and decreasing the levels of photo-protective pigment β-carotene. This in turn minimized the negative significances of larger cell volume-induced package effects. Exposure to extreme conditions such as photoinhibitory light levels, UVA and UVB radiation, and nutrient and temperature stress could result in oxidative damage to cellular components especially proteins. These kinds of damage result in breakdown of the damage component and re-synthesis of these components or adaptive changes to the cellular components. These changes however have a cost in terms of energy (i.e., ATP, NADPH) and resources which could have been directed towards biomass and lipid synthesis. Protein turnover during scotophase has been shown to consume about one-third of the total respiratory ATP production [33]. Exposure to nitrogen-limiting conditions could result in upregulation of nitrogenase in diazotrophic cyanobacteria, which in turn could result in 10% decrease in growth rates [34, 35]. These are avoidable energy and resource expenditure, which can be easily prevented by providing optimal light, nutrient, and temperature during mass cultivation.

Lipids are not secreted/excreted actively by all microalgae, with exception of *Botryococcus* sp. [36]. This constrains the maximum amount of lipid that is synthesized and stored in a microalgal cells, as the largest reported cell volume for a microalgae is for species *Noctiluca scintillans* belonging to the class Dinophyceae (83,700,000 μm3 ) [37]. The most commonly used microalgal species in biotechnology belong to the class Chlorophyceae such as *Chlorella* sp., *Haematococcus* sp., and *Scenedesmus* sp., which have cell volumes of 34.5, 48, and 26,200 μm3 , respectively [38], and the species belonging to class Bacillariophyceae such as *Phaeodactylum tricornutum* and *Thalassiosira pseudonana* which have cell volumes of ~60–100 μm3 and 28–200 μm3 [37, 39]. On the other hand, the maximum cellular density has an inverse relationship with the volume of a cell, by a factor of 3/4 [38]. This further constrains the maximum amount of lipids that can be synthesized in a microalgal mass culture. However, if the lipids were secreted in a manner similar to EPS, physiological limitations such as cell volume or maximum density would not play a constraining role on the maximum amount of lipid that can be synthesized in an algal mass culture. Microalgal species like *Botryococcus* sp. has already been shown to actively secrete lipids that can be converted into fuel-grade biodiesel. However, their slower growth rates limit the maximum lipids that can be synthesized. Microalgal species like *Scenedesmus* has been shown to grow as fast as 1.53 per day [40]. Therefore, genetic engineering techniques to integrate the lipid-secreting trait into the fast-growing *Scenedesmus* sp. could be a possible solution.

Phototrophic modes of cultivation depend on two critical factors, namely, light and carbon dioxide [16]. Therefore, microalgae grown phototrophically can either be limited in light or carbon dioxide or both, limiting the maximum cellular density one can achieve using this mode of cultivation. In addition, various inefficiencies described above with light-harvesting abilities of microalgae, lower affinity for CO2 of RUBISCO, cost associated with protein turnover, and photoinhibition can be simply avoided by growing microalgae heterotrophically and mixotrophically. Extensive amount of research suggests enhanced biomass production under heterotrophic and mixotrophic modes of cultivation, increasing the cellular density to as much as 4 to 5 times [41, 42]. Further modification in these modes of cultivation such as using

fed-batch mode has resulted in further increase in biomass as much as two- to fivefold [43, 44]. Therefore, by growing a hybrid strain of lipid-secreting *Botryococcus* sp. and fast-growing *Scenedesmus* sp., in a fed-batch heterotrophic or mixotrophic cultivation system, one can possibly overcome the physiological limitations of the maximum amount of lipids that can be synthesized in a microalgal system.
