2. Microalgae metabolism and mixotrophic growth kinetics

Biomass and product productivity are significantly affected by the culture condition; energy and carbon supply impacts directly biomass and product concentration. In effect, different metabolic growth modes for microalgae have been recognized: (a) autotrophy, in which light is the sole source of energy and inorganic carbon is the sole source of carbon; (b) heterotrophy, in which energy and carbon are both obtained exclusively from an organic carbon source, such as glucose, glycerol, and acetate, and growth can proceed without light supply; (c) mixotrophy, in which the photosynthetic microorganisms obtain energy from light and organic carbon sources and carbon is obtained from organic and inorganic carbon sources [4, 5]; and (d) photoheterotrophy, in which carbon can be obtained from organic compound but strictly with a light supply [5]. Chojnacka and Noworyta designed an empirical mathematical model to describe mixotrophic growth; in this model heterotrophic and autotrophic cultures are fractions of mixotrophic growth, but the metabolic interaction of photosynthesis and heterotrophy is important to improve biomass density and consequently secondary metabolite productivity [6].

Light as a source of energy for photosynthetic organisms is the main limiting factor during cultivation process of these organisms. In light intensities above the light saturation point, photosynthesis rate is directly proportional to the incident light supplied. The photosynthetic system of many microalgae becomes saturated to a radiation close to 30% of the total solar irradiance, i.e., between 1700 and 2000 μEm<sup>2</sup> s 1 . Some species of phytoplankton grow to optimal intensities of 50 μEm<sup>2</sup> s <sup>1</sup> and are photoinhibited at around 130 μEm<sup>2</sup> s 1 . The culture limitation by photoinhibition is the most important problem for commercial cultivation of microalgae. A possible solution is to assume that the heterotrophic metabolism in photosynthetic cells occurs, replacing or supplementing energy and carbon requirements from organic sources. Some studies suggest that mixotrophic, autotrophic, and heterotrophic metabolic activities occur simultaneously during cell growth [7]. The relative contribution of autotrophy to biomass production increases by increasing the light supply coefficient (kJ kg m<sup>2</sup> s 1 ) or with an increase in the supply of CO2 and a decrease of organic carbon source supply. For example, at a light supply coefficient of 0.5 at 0.03 and 10% of CO2 concentration, the ratio of contribution of autotrophy (heterotrophy/autotrophy) to the biomass production was of 98:2 and 70:30, respectively [8]. A respirometric procedure has been proposed to obtain half saturation constant values for several nutrients; it is useful for modeling bioprocess for photosynthetic microorganisms [9]. These methods can be useful to evaluate organic substrates to be used in cultures, in a practical way.

### 2.1 Contribution of autotrophic and heterotrophic metabolisms during the mixotrophic cultivation performance

Prior works [8, 10] conducted a detailed analysis of the heterotrophic and autotrophic modes simultaneously in Euglena gracilis and Spirulina platensis,

(1) the culture medium design—it is necessary to fix the nutrient composition to provide the right source of carbon and energy depending on the microalgae strain and secondary metabolite to be produced; (2) reducing adverse conditions for culture, such as oxygen accumulation, CO2 efficient supply, and sufficient light distribution. For this purpose, studies on the photobioreactor prototype should be performed; (3) once that photobioreactor prototype works well, critical factor

Biomass and product productivity are significantly affected by the culture condition; energy and carbon supply impacts directly biomass and product concentration. In effect, different metabolic growth modes for microalgae have been recognized: (a) autotrophy, in which light is the sole source of energy and inorganic carbon is the sole source of carbon; (b) heterotrophy, in which energy and carbon are both obtained exclusively from an organic carbon source, such as glu-

(c) mixotrophy, in which the photosynthetic microorganisms obtain energy from light and organic carbon sources and carbon is obtained from organic and inorganic carbon sources [4, 5]; and (d) photoheterotrophy, in which carbon can be obtained

heterotrophy is important to improve biomass density and consequently secondary

Light as a source of energy for photosynthetic organisms is the main limiting factor during cultivation process of these organisms. In light intensities above the light saturation point, photosynthesis rate is directly proportional to the incident light supplied. The photosynthetic system of many microalgae becomes saturated to

tion by photoinhibition is the most important problem for commercial cultivation of microalgae. A possible solution is to assume that the heterotrophic metabolism in

supply of CO2 and a decrease of organic carbon source supply. For example, at a light supply coefficient of 0.5 at 0.03 and 10% of CO2 concentration, the ratio of contribution of autotrophy (heterotrophy/autotrophy) to the biomass production was of 98:2 and 70:30, respectively [8]. A respirometric procedure has been proposed to obtain half saturation constant values for several nutrients; it is useful for modeling bioprocess for photosynthetic microorganisms [9]. These methods can be useful to evaluate organic substrates to be used in cultures, in a practical way.

2.1 Contribution of autotrophic and heterotrophic metabolisms during the

Prior works [8, 10] conducted a detailed analysis of the heterotrophic and autotrophic modes simultaneously in Euglena gracilis and Spirulina platensis,

. Some species of phytoplankton grow to optimal intensities of

1

1

. The culture limita-

) or with an increase in the

criteria for scale-up bioengineering process should be fixed [1–3].

Microalgae - From Physiology to Application

2. Microalgae metabolism and mixotrophic growth kinetics

cose, glycerol, and acetate, and growth can proceed without light supply;

from organic compound but strictly with a light supply [5]. Chojnacka and Noworyta designed an empirical mathematical model to describe mixotrophic growth; in this model heterotrophic and autotrophic cultures are fractions of mixotrophic growth, but the metabolic interaction of photosynthesis and

a radiation close to 30% of the total solar irradiance, i.e., between 1700 and

photosynthetic cells occurs, replacing or supplementing energy and carbon requirements from organic sources. Some studies suggest that mixotrophic, autotrophic, and heterotrophic metabolic activities occur simultaneously during cell growth [7]. The relative contribution of autotrophy to biomass production increases

by increasing the light supply coefficient (kJ kg m<sup>2</sup> s

mixotrophic cultivation performance

<sup>1</sup> and are photoinhibited at around 130 μEm<sup>2</sup> s

metabolite productivity [6].

1

2000 μEm<sup>2</sup> s

50 μEm<sup>2</sup> s

194

respectively, under strict control of culture conditions; mixotrophic biomass concentration and growth rates resulted in the sum of the biomass or growth rates of heterotrophic and autotrophic cells in growing culture in parallel. The yields produced approximately the same amount of biomass produced in mixotrophic conditions; a mathematical approach can be summarized in Eqs. (1)–(8) [8, 10]. Assuming that mixotrophic growth is derived from cells growing in autotrophic and heterotrophic conditions, it can be described mathematically by defining the contribution of both metabolic growth modes. This can be simplified in Eq. (1):

$$X\_M = X\_A + X\_H \tag{1}$$

Supposing that α is the heterotrophic fraction in mixotrophic growth, XH (biomass from heterotrophy) may be expressed as follows:

$$X\_H = aX\_M \text{ with the condition } 0 < a < 1\tag{2}$$

Combining Eqs. (1) and (2), biomass in mixotrophic (XM) conditions can be expressed as follows:

$$X\_M = \frac{X\_A}{1 - a} \tag{3}$$

in which XA is the biomass from autotrophy; the growth rate ratio can be expressed as follows:

$$\mathbf{1} \approx \left[ \frac{d\mathbf{X}\_{\mathcal{M}}/\_{\mathrm{dt}}}{d\mathbf{X}\_{\mathcal{A}}/\_{\mathrm{dt}} + d\mathbf{X}\_{\mathcal{H}}/\_{\mathrm{dt}}} \right] \mathrm{(g \; d^{-1})} \tag{4}$$

The α coefficient is low when the incident light reaches all cells in the photobioreactor, where cell density should be low enough to avoid hiding among cells. Once that cell density increases, heterotrophic fraction increases as well, in the presence of an organic substrate as carbon source. To estimate α at any time, the value of α can be represented by an α<sup>i</sup> which can be constant for a period of time, Δti.

Then, Eq. (4) can be expressed as follows:

$$\frac{\Delta X\_M}{\Delta t\_i} = \frac{1}{1 - a\_i} \frac{\Delta X\_A}{\Delta t\_i} \tag{5}$$

By replacing in Eq. (5) of autotrophic growth, the equation included incident light:

$$\frac{\Delta X\_M}{\Delta t\_i} = \frac{1}{1 - a\_i} K l\_0 \tag{6}$$

where <sup>K</sup> <sup>¼</sup> YkJA V .

Integrating Eq. (6) results in

$$\int\_{X\_{Mi}}^{X\_{Mi+1}} \Delta X\_{M=} \frac{Kl\_o}{1 - \alpha\_i} \int\_{t\_i}^{t\_{i+1}} \Delta t\_i \tag{7}$$

And then, α<sup>i</sup> at any time can be calculated:

$$\alpha\_{i} = 1 - \frac{Kl\_{o}}{X\_{M\_{i+1}} - X\_{M\_{i}}} (t\_{i+1} - t\_{i}) \tag{8}$$

With Eq. (8), α value was 0.02 at the beginning of culture, independent of incident light, to 0.61 after several hours of cultivation. Carbon balance showed inorganic carbon and carbon from glucose were consumed simultaneously; CO2 produced from respiration was used as carbon source during autotrophic growth [11]. Ogbonna and McHenry observed similar behavior in Euglena gracilis. Heterotrophic and autotrophic growth occurred simultaneously and independently [8]; this work defined two fractions instead of one as follows:

$$\frac{dX\_M}{dt} = \frac{dX\_A}{dt} + \frac{dX\_H}{dt} \tag{9}$$

disadvantages of open systems are the significant loss of water by evaporation, the loss of CO2 into the atmosphere, the pollution, and the need for considerable surface for cultivation. Since the 1990s, in certain parameters such as the selection of species with efficient incident light utilization, the path of the incident light through the photobioreactor (PBR), the thickness of the wall, the mixing regime, and release of O2 via degassing, CO2 supply, have been focus on several developments [12]. Closed or semi-closed PBRs, based on different design concepts, have been

implemented and tested at a pilot level. The latest developments seem to be directed toward tubular or plate-type compact configurations as well as combinations of these major designs in the form of distributing light over an expanded surface [13].

Microalgae need enough quality and quantity of light supply, and it should be taken into account as a primary critic factor to design proper PBR. Cell density can

tion of the distance among cells over 250 times, and the cell size can reduce its size 10 times as well. By improving mix capabilities of the PBR, hydrodynamic shearing stress over the cells can be increased; also, it can reduce growth or even cell death at high stress conditions [14]. The temperature has a greater influence on respiration and photorespiration than photosynthesis; when CO2 or light is limiting for photosynthesis, the influence of temperature is negligible. In contrast, an increase in the temperature will increase significantly the respiration, but flow of carbon through the Calvin cycle increases marginally. In other words, the net efficiency of photosynthesis declines at high temperatures. This effect can worsen in culture suspension by the difference in the solubility of CO2 and O2 at high temperatures. Normal temperatures for the growth of microalgae ranged between 25 and 30°C; an increment in the temperature affects the lipid production; at higher temperatures saturated free fatty acids are produced, while low temperatures favor unsaturated free fatty acid formation [15]. High concentration of O2 can build up in

closed PBR; if this happens photosynthesis can be damaged by decreasing

in the PBR, cell growth, and secondary metabolite production.

microalgae growth, and an improvement in the PBR should be implemented as an

The first generation of closed PBR finds limitations over 50–100 L of culture volume; this was not effective for light supply to produce higher biomass density. Several designs of light distribution over the PBR, mainly underwater lamps, optical fiber, and column-shaped photobioreactors, have been used to provide an efficient production system; however, not much success has been obtained [12]. This is the main challenge in the future to find the appropriate scaling criteria for a larger irradiate surface, mass transfer, and coupled steps upstream and downstream processes [17]. The difficulty to scale up PBRs is to establish the inherent relationship among physical parameters involved in the design and the physiology of the microalgae to be cultured. An important design rule is to define quantitatively parameters to describe the interactions between incident light, the light distribution

To encourage the use of microalgae, it is necessary to implement a step-by-step system at different levels. The first step is the bioprospecting for selecting the most

; it produces a reduc-

4. Main problems in closed photobioreactors: light supply,

increase from 10<sup>3</sup> cells ml<sup>1</sup> to densities above 10<sup>8</sup> cells ml<sup>1</sup>

temperature, and oxygen accumulation

Microalgae Cultivation for Secondary Metabolite Production

DOI: http://dx.doi.org/10.5772/intechopen.88531

effective gas exchange [16].

197

5. Photobioreactor design and scale-up

The mixotrophic growth rate dXM/dt was equal to the sum of the heterotrophic growth rate dXH/dt and the rate of autotrophic growth dXA/dt. However, when two metabolic activities interact and when the presence of an organic carbon source affects the autotrophic metabolism or when the light affects the heterotrophic metabolic activity, then the heterotrophic rate in the mathematical description can be modified in accordance with Eq. (10), where dXM/dt is the total mixotrophic growth rate and β and α are coefficients of the autotrophic and heterotrophic fractions of the total mixotrophic growth rate, respectively:

$$\frac{dX\_M}{dt} = \beta \frac{dX\_A}{dt} + a \frac{dX\_H}{dt} \tag{10}$$

The values of α and β can be calculated on the basis of dXA/dt autotrophic growth rate and dXH/dt heterotrophic growth rate, both during mixotrophic culture.

$$\beta = \frac{d\mathbf{X}\_{\mathbf{A}}/\_{\text{dt}}}{d\mathbf{X}\_{\mathbf{M}}/\_{\text{dt}}} \tag{11}$$

$$\alpha = \frac{d\mathbf{X}\_{\text{fl}}/\_{\text{dt}}}{d\mathbf{X}\_{\text{M}}/\_{\text{dt}}} \tag{12}$$

The sum of the values of β and α is 1.0 when the growth proceeds independently and simultaneously; when the sum is more than 1.0, there is an effect of promotion; and when it is <1.0, there is an inhibitory effect. It is clear that when mixotrophic growth occurs, both growth modes, namely, autotrophic and heterotrophic growth, have contribution in the final biomass and metabolite production; Eqs. (1)–(12) may be used to describe mathematically the secondary metabolite formation. An important stoichiometric relationship exists on carbon metabolism, with the pH changes driven by consumption of carbon source. Bicarbonate consumption increases pH, whereas glucose consumption decreases the pH due to CO2 production, being the reason that pH is kept almost constant during mixotrophic growth, CO2 consumption by photosynthesis, this balance may reflect the type of predominant metabolism.

#### 3. Photobioreactor systems: open and closed systems

Despite certain variability in the shape of open and closed systems, technical designs for open systems are the type race track, moved by paddles, usually operating at depths of 15–20 cm. At this depth, the growth rate of microalgae can be 15 g m�<sup>2</sup> d�<sup>1</sup> , with a lipid content of 25%. Similar designs in terms of operation are the circular ponds, which are commonly found in Asia and Ukraine [3]. The major Microalgae Cultivation for Secondary Metabolite Production DOI: http://dx.doi.org/10.5772/intechopen.88531

With Eq. (8), α value was 0.02 at the beginning of culture, independent of incident light, to 0.61 after several hours of cultivation. Carbon balance showed inorganic carbon and carbon from glucose were consumed simultaneously; CO2 produced from respiration was used as carbon source during autotrophic growth [11]. Ogbonna and McHenry observed similar behavior in Euglena gracilis. Heterotrophic and autotrophic growth occurred simultaneously and independently [8];

dt þ

The mixotrophic growth rate dXM/dt was equal to the sum of the heterotrophic growth rate dXH/dt and the rate of autotrophic growth dXA/dt. However, when two metabolic activities interact and when the presence of an organic carbon source affects the autotrophic metabolism or when the light affects the heterotrophic metabolic activity, then the heterotrophic rate in the mathematical description can be modified in accordance with Eq. (10), where dXM/dt is the total mixotrophic growth rate and β and α are coefficients of the autotrophic and heterotrophic

dXH

dt (9)

dt (10)

(11)

(12)

this work defined two fractions instead of one as follows:

Microalgae - From Physiology to Application

fractions of the total mixotrophic growth rate, respectively:

culture.

predominant metabolism.

15 g m�<sup>2</sup> d�<sup>1</sup>

196

dXM

dt <sup>¼</sup> <sup>β</sup> dXA

The values of α and β can be calculated on the basis of dXA/dt autotrophic growth rate and dXH/dt heterotrophic growth rate, both during mixotrophic

> <sup>β</sup> <sup>¼</sup> dXA=dt dXM=dt

> <sup>α</sup> <sup>¼</sup> dXH=dt dXM=dt

production, being the reason that pH is kept almost constant during mixotrophic growth, CO2 consumption by photosynthesis, this balance may reflect the type of

Despite certain variability in the shape of open and closed systems, technical designs for open systems are the type race track, moved by paddles, usually operating at depths of 15–20 cm. At this depth, the growth rate of microalgae can be

the circular ponds, which are commonly found in Asia and Ukraine [3]. The major

, with a lipid content of 25%. Similar designs in terms of operation are

3. Photobioreactor systems: open and closed systems

The sum of the values of β and α is 1.0 when the growth proceeds independently and simultaneously; when the sum is more than 1.0, there is an effect of promotion; and when it is <1.0, there is an inhibitory effect. It is clear that when mixotrophic growth occurs, both growth modes, namely, autotrophic and heterotrophic growth, have contribution in the final biomass and metabolite production; Eqs. (1)–(12) may be used to describe mathematically the secondary metabolite formation. An important stoichiometric relationship exists on carbon metabolism, with the pH changes driven by consumption of carbon source. Bicarbonate consumption increases pH, whereas glucose consumption decreases the pH due to CO2

dt <sup>þ</sup> <sup>α</sup> dXH

dXM dt <sup>¼</sup> dXA disadvantages of open systems are the significant loss of water by evaporation, the loss of CO2 into the atmosphere, the pollution, and the need for considerable surface for cultivation. Since the 1990s, in certain parameters such as the selection of species with efficient incident light utilization, the path of the incident light through the photobioreactor (PBR), the thickness of the wall, the mixing regime, and release of O2 via degassing, CO2 supply, have been focus on several developments [12]. Closed or semi-closed PBRs, based on different design concepts, have been implemented and tested at a pilot level. The latest developments seem to be directed toward tubular or plate-type compact configurations as well as combinations of these major designs in the form of distributing light over an expanded surface [13].
