Keywords

4.1 Introduction

Microalgae are photosynthesizing microorganisms; they may be prokaryotic or eukaryotic in nature, present in aquatic areas like ponds, lakes, streams, oceans, deep sea waters, soil, and on earth. They are characterized by rapid growth even at high pressures (deep seas), in acid environments, and in extreme conditions. The microalgae play a critical role in CO2 sequestration, bioactive compounds, and biofuel production, involving in wastewater treatment, and needed a potential research for the enhancement of the end products (Guzzon et al. 2008; Kumar et al. 2009). Therefore, a critical research is needed to achieve full-scale and continuous process including (1) assess nutrient uptake, (2) improved algae growth, (3) wastewater utilization, (4) CO2 sequestration efficiency, (5) improved algal harvesting, (6) enhanced extraction and purification, and (7) economic considerations (Pulz 2001; Carvalho and Malcata 2001).

4.2 Types of Microalgae

Microalgae are observed as single cells to colonies (a group of cells) and also as filamentous eukaryotic structures (chain like multiple cells), from prokaryotes to eukaryotes, and are classified according to biological evolution upon the time from cyanoprokaryotes to eukaryotes. Cyanoprokaryotes are with no distinct nucleus and chloroplasts (primitive of life), whereas eukaryotes are having well-defined cell wall, nucleus, and other cellular organelles. Further in algae vascular tissues, embryo and sex organs are absent like higher plants. Eukaryotic algae are classified into several divisions, i.e. Chrysophyceae, Chlorophyceae, Euglenaphyceae, Cyanophyceae, Rhodophyceae, Pyrrophyceae, Phaeophyceae, and Xanthophyceae. Further, general classification depends on the pigments present, such as green (chlorophyll), blue (phycocyanin), yellow (xanthophylls), orange (carotene), red (phycoerythrin), and brown (fucoxanthin) algae (Frac et al. 2010). The major differences in characteristics and the classification of algae are summarized in Table 4.1.

Table 4.1 Major differences in characteristics present in various divisions of algae
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4.3 Components Present in Algae

The major components of microalgae are very rich in lipids, carbohydrates, proteins, and also vitamins, pigments, etc. in various percentages of their dry weight, and these percentages may vary depending upon the variety of microalgae. Among them largest fractions of lipids and proteins are present, lipids are used for production of biofuels and edible oils (Delrue et al. 2012). In case of proteins and carbohydrates, they are used for the protein supplements and polymers manufacturing, respectively (Tables 4.2 and 4.3).

Table 4.2 Various components present in microalgae as percentages of dry weight
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Table 4.3 Various components present in microalgae as percentages of dry weight
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4.4 Cultivation of Microalgae

Microalgae are generally unicellular autotrophs/heterotrophs which convert inorganic compounds in the presence of light energy into organic compounds. The earliest photoautotroph organisms (anoxygenic photosynthetic process) utilize light source to extract the protons and electrons from donors (Fe2+, H2S, etc.), and reduction of CO2 ultimately produces organic compounds. Blue-green algae (cyanobacteria) are prokaryotic cells containing DNA in nucleoplasm and peripheral region (Chromoplast) covered with photosynthetic sheets in parallel and closed to cell surface. Eukaryotic algae (microalgae) have special organelle chloroplasts with alternative layers of thylakoids and stroma which is involved in photosynthetic process. Microalgae have the potential for CO2 fixation, biofuel, and bioactive compounds production due their capability of photosynthesis which transforms CO2 in to biomass and into several value-added products. So, microalgae cultivation is an option to reduce CO2 release from the fossil fuel combustion.

During photosynthesis, light energy–controlled oxidoreduction reaction yielded by chlorophyll in which CO2 and H2O are transformed into oxygen and sugars in two stages, i.e., light and dark reactions. In thylakoid membranes, the light power is transformed into chemical force, facilitating a biochemical reductant NADPH2 and ATP. In stroma, NADPH2 and ATP are utilized for consecutive biochemical reduction of CO2 to sugars (Fig. 4.1). Light energy is available as photons (quanta) and as a product of plank’s constant and its frequency (E = hν). The light energy incident on the surface was measured as radian flux energy; the units are power per area (W m−2 or Jm−2 s−1). In the process of photosynthesis, photon flux density is considered as the quantity of photons (quanta) that are accrued on the surface per unit time which is measured in μmol quanta m−2 s−1 or μEm−2 s−1.

Fig. 4.1
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Photosynthesis in microalgae with both light and dark reactions

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4.5 Nutritional Requirements of Algae Growth

Algae are categorized as autotrophs and heterotrophs and, depending upon energy and carbon sources, are categorized as photoautotrophs, chemoauthotrophs, chemoheterotrophs, phagocytosis. Further due to mixed characteristics, they are again subdivided into photoheterotrophs, auxotrophs, and mixotrophs. The autotrophic algae depend on light for the reduction of CO2 and water by the release of O2. Photo-autotrophs need only inorganic minerals for their growth, whereas some organic combinations are also required for growth (e.g., vitamins). Vonshak (1986) summarized the nutritional necessities for the fabrication of algal recipes, i.e., (a) the total salt contents according to the habitat from where the algae originate, (b) the main ionic contents such as K+, Mg2+, Na+, Ca2+, SO4=, and Cl−, (c) the nitrogen source, (d) carbon source either CO2 or HCO3−, (e) pH, (f) minor elements and chelating agents, and (g) vitamins. Carbon (C), nitrogen (N), and phosphorus (P) are the three main essential nutrients for autotroph growth, and their supply is essential for the effective microalgae growth. The nutrient-rich supply could effectively impacted on the algal cultivation process for optimal biomass production.

4.5.1 Carbon

Carbon is an essential element as sole feedstock to form the carbon skeleton in the microalgae biomass growth and must available in the form of inorganic or organic source enriched medium. For high growth rate of microalgae, CO2 or HCO3− are very important in the medium as it is maintained the pH like as buffer system in the liquid medium that is optimal for mass cultivation. The buffering system chemically expressed as CO2 – H2CO3 –HCO3−– CO32− is having greater importance in maintenance of the pH in cultivation of microalgae. On the other hand, glycylglycine and Tris(2-amino-2-[hydroxymethyl]-1–3-propanediol) are frequently preferred as buffering system. Chemically the buffering system involving photosynthesis process to produce CO2 is as following:

$$ 2{{\mathrm{H}\mathrm{CO}}_3}^{-}\leftrightarrow {{\mathrm{CO}}_3}^{2-}+{\mathrm{H}}_2\mathrm{O}+{\mathrm{CO}}_2 $$
(4.1)
$$ {{\mathrm{HCO}}_3}^{-}\leftrightarrow {\mathrm{CO}}_2+{\mathrm{OH}}^{-} $$
(4.2)
$$ {{\mathrm{CO}}_3}^{2-}+{\mathrm{H}}_2\mathrm{O}\leftrightarrow {\mathrm{CO}}_2+2{\mathrm{OH}}^{-} $$
(4.3)

The above reactions shows that OH− accumulation leads to rise in pH during photosynthetic CO2 fixation. This is natural phenomenon to rise in pH upto “11” and increased density of algal growth without additional supply of CO2. One can control the pH rise to “11” by additionally sparging CO2 into the growth medium; it ultimately leads to highly dense algal growth. Further, as discussed above, the carbon sources provide organic acids, alcohols, and sugars for mixotrophic algal cultivation.

4.5.2 Nitrogen

Nitrogen is an important element and occupies >10% of dry weight of microalgae and helps in biomass production. Nitrogen could be supplied as ammonia (NH4+), urea, and nitrate (NO3−) to the growth medium in mass cultures. Ammonia is useful in pH maintenance in case of additional supply of CO2, but here the mechanism is releasing of protons (H+). To achieve higher yields of microalgae biomass production, adequate supply of nitrogen is needed. Few cynobacterial species are capable of reducing the atmospheric elemental N2 to NH4+ by nitrogenase enzyme. In the efficient production of biofuels by microalgae, in the microalgae medium ingredients few nutrients are rationalized or limited in quantity. In this regard, nitrogen is limited supply material (nitrogen starvation) in the medium leads to accumulation of lipids reaching to higher levels in microalgal biomass. In nitrogen starvation the microalgae synthesize the TG (triglyceride) and utilized to storage of energy and carbon. These stored TG-FA have undergone transesterification process and release methyl and ethyl esters. Nitrogen scarcity can lead to the accumulation of lipids, but it compromises the cell growth (Chen et al. 2017). To avoid it in the first stage of growth, rich supply of nitrogen is needed; in the later stage, nitrogen scarcity helps to build up lipids in biomass for biofuel production as good feedstock.

4.5.3 Phosphorus

This is a critical element for algae growth, energy transfer, and also in the biosynthesis of nucleic acids, adenosine triphosphate, phospholipids, etc. Algal biomass contains >1% of phosphorus, and it is easily bound with other ions (iron, CO32−) which leads to precipitation, resulting in unavailability of phosphorus to algal uptake. In the growth medium, the preferred form of phosphorus is orthophosphate, and the uptake is energy dependent. Adenosine triphosphate is the preliminary product of photosynthesis of algae, so phosphorus is essential for achieving higher yields of microalgae production. Phosphorus limitation or scarcity has no superior effects on microalgae growth as nitrogen scarcity (Kamalanathan et al. 2015). Therefore, phosphorus supply is sufficiently needed (Chu et al. 2013) to attain effective density of microalgae for the biofuel production at the first stage, and then nitrogen scarcity is required at the second stage.

4.5.4 Macro- and Micronutrients

Essential nutrients required except phosphorus, nitrogen, and carbon are 30 elements in the form of minerals of high quantity (macronutrients) and in trace amounts (micronutrients) for microalgae growth. Macronutrients are Ca, Mg, S, K, Na, Fe, etc. and micronutrients are enlisted as Cu, Mn, B, Zn, Co, Mo, V, Se, etc. Calcium is an important element in cell wall and membrane structures and performs as intracellular messenger that harmonizes response toward environmental changes. Magnesium as part of enzymes is directly related to photosynthesis and CO2 fixation. Magnesium supplementation in the culture media could increase the photosynthesis activation, thereby increases the accumulation of lipids in biomass. Esakkimuthu et al. (2016) explained in their work on the starvation of calcium and phosphorus that decrease in lipid content in microalgae and they suggested as lower amount of calcium must be provided than the starvation. In modern cultivation of algae primarily provided all nutrients richly and later stages starvation of nitrogen, little amount of calcium could be preferred to achieve the higher yields of biomass and optimized production of biofuels. Salinity of the growth medium could be maintained by the addition of salts, i.e., NaCl, KCl, MgCl2, CaCl2, NaCl, and KCl are used as salinity modifiers to increase the external osmotic pressure of the cell which may affect the fluidity and permeability of the membrane. MgCl2 and CaCl2 are not modifying the salinity of the medium efficiently than the above salts but involving in other regulations. Sajjadi et al. (2018) revealed that the when high salinity in the medium, the osmo-regulation mechanism is started in the cell to equilibrate external salinity condition. This equilibration and osmoregulation could induce fatty acid metabolism which leads to accumulation of lipids. Silicon is also an important constituent which is involved in the formation of cell wall especially in diatoms. Cobalt is essential for the synthesis of vitamin B12, and other minor nutrients are very important as they are cofactors with the enzymes and involved in the metabolism. Metal chelators (EDTA) are required to avoid phosphorus precipitation in the medium and avoid the unavailability of phosphorus which is essential for cellular structures and division of microalgal cells. So, the macro and micro inorganic nutrients are most necessary for microalgae-augmented biomass harvest and biofuel production. Several microalgae media available (55 types) in the literature are depicted in the Tables 4.4, 4.5, 4.6, 4.7, 4.8, and 4.9 along with their ingredients, quantities and compared with conventional medium. However, here the components, quantities, and suitability of the medium for microalgae cultivation are considered by diversity of algae and target of the product to be achieved.

Table 4.4 Various culture media and their compositions (g L−1 or otherwise stated)
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Table 4.5 Various culture media and their compositions (g L−1 or otherwise stated)
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Table 4.6 Various culture media and their compositions (g L−1 or otherwise stated)
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Table 4.7 Various culture media and their compositions (g L−1 or otherwise stated)
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Table 4.8 Various culture media and their compositions (g L−1 or otherwise stated)
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Table 4.9 Various culture media and their compositions (g L−1 or otherwise stated)
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4.5.5 Other Considerations

Along with the above conditions, many other parameters will influence the effective cultivation of microalgae. They are (1) temperature, (2) light, (3) pH, (4) CO2, (5) salinity, (6) O2, (7) mineral bioavailability, (8) carbon bioavailability and cellular characteristics, (9) growth inhibition, (10) cell density, and (11) cell fragility. Reactor characteristics affecting the microalgae growth are (1) rate of mixing, (2) gas bubble size, (3) gas bubble distribution, (4) fluid dynamics, (5) gas exchange capacity, (6) mass transfer, (7) dilution rate, (8) light intensity, (9) culture depth, and (10) maintenance of dark spots. Further, the cultural growth characteristics also influence the growth of microalgae, i.e., (1) toxic chemicals to microalgae, (2) pathogens to microalgae, (3) competitive growth of other algae, (4) management of photo inhibition, (5) management of photorespiration, and (6) yield frequency of microalgae.

In the designing of the microalgae medium, the nutritional ingredients and their quantities are consider for the enhanced yields of specific metabolites. In few instances, it is essential to alter the quantities and amounts of one or more nourishments (Shin et al. 2018). So, one must know the definite features of each and every group of algae, their physiology, nourishment prerequisites, growth circumstances, and specific considerations to alter the composition of medium (Milano et al. 2016). Cuellar García et al. (2019) evaluated various culture mediums on two microalgae S. obliquus and C. vulgaris for biodiesel productivity. They used Bristol, Bold, and AAP (modified algal assay procedure) culture conditions and studied about which one is the best in favor of biomass growth for lipid accretion. The results have been revealed that the usage of Bold medium and AAP medium for S. obliquus and C. vulgaris respectively, showed higher production of biomass containing more lipids when compared with other media. BG 11 medium is suggested for freshwater and soil Cyanophyceae, diatom medium is recommended for freshwater Bacillariophyceae algae, and DY-III medium is the best for freshwater Chrysophyceae group of algae. Bold Basal Medium (BBM) is used broadly for the freshwater Chlorophyceae, Xanthophyceae, Chrysophyceae, and Cyanophyceae. AF-6 medium is useful for algae cultivation where slight acidic condition is required, i.e., Euglenophyceae, xanthophytes, dinoflagellate, green ciliate, volvocalean algae, and many cryptophytes. AK medium will be used for the cultivation of almost all varieties of marine algae. Both “F” medium and “G” medium are useful for marine algae cultivation in salt as well as brackish waters. Both “K” medium and “LO” medium are useful for marine microalgae, especially oligotrophic salt and brackish waters. Combo medium is used broadly for the cultivation of cryptophytes, cyanobacteria, diatoms, and green algae, whereas MNK medium is the best suitable for especially marine microalgae, coccolithophores.

Sueoka’s medium is especially used for the cultivation of Chlorophyceae family algae. In the similar manner, MES and Jaworski’s media are used broadly for pure water microalgae cultivation. Walne’s culture medium is broadly used for marine microalgae principally set in commercial culturing. ASN-III, PCR-S11, and Chu-II media are most preferably recommended for the Cyanobacterial strain cultivation. Botryococcus braunii is a colonial microalgae cultivated in modified Chu 13 medium for the production of lipids (Yamaguchi et al. 1987). F/2 medium and ESAW medium are used for the broad spectrum for oligotrophic marine algae and coastal open ocean algae cultivation. CY-II, Zarrouk’s, and Johnson’s media were used to cultivate Cyanophora paradoxa, Arthrospira sp., and Dunaliella sp. (halophilic algae), respectively. Chlorella vulgaris is studied with Chu medium, and it is found that hydrocarbons (HC), free fatty acids, sterol esters (SE), acetone mobile polar lipids, aliphatic alcohols (ALC), and phospholipids (PL) yield are more. Debjani et al. (2012) experimented on different media, i.e., dry-grind ethanol thin stillage (TS), soy whey (SW), and modified basal medium (MBM) as media for the cultivation of C. vulgaris. After 4 days of incubation, the biomass yield was 9.8, 6.3, and 8.0 g L−1 and the oil content was 43, 11, and 27% (w/w) in the media TS, SW, and MBM, respectively. The quality of fatty acid profile is also tested as linoleic and linolenic forms, the oil is richer and good quality when grown in TS and MBM media. Xiong et al. (2008) reported on the cultivation of C. protothecoides to achieve more dense biomass in the production of biodiesel using Wu’s culture medium successfully.

As reported by Richmond and Becker (1986), the higher yields of algal biomass could be achieved when (a) providing nutrition-rich medium, (b) positive environmental parameters, and (c) turbulent flow mixing. The turbulent flow mixing enhances the diffusion rate of CO2, exchange rate of nutrients in betwixt the algal cells, and fluid medium; further augmented light/dark periods lead to efficient photosynthesis and productivity (Grobbelaar 1994). Microalgae is small in size and available in liquid medium as suspension offers direct contact with nutrients present in medium. Upon the sparging of air due to higher surface area available on algae, CO2 fixation and also solar energy transmission rate are high. The main advantage of microalgae cultivation is that the maximum annual biomass productivity is more, due to nutrients, solar light, adsorption of CO2, and high surface area availability on algae. High surface area allows the easy exchange of gases, nutrients, and solar energy which leads to faster growth than other sources of biofuels.

4.6 Bioreactors for Microalgae Cultivation

Since microalgae could grow well at immense biodiversity, possibility to use waste water as nutritional medium, can sequester CO2; producce O2 and can produce high cell density are great features so that leads to much attention by scientists and considered as microalgae as feedstock for biofuels production. Accurate fabrication, design of reactors, and optimization of process by biotechnology of microalgae facilitate the achievement of augmented cell density mass cultures. The overall knowledge is required to scale up the operational parameters, i.e. (1) light energy, (2) mass transfer, (3) shear forces, and (4) rate of mixing. The above-mentioned variables are strongly interconnected and decide the yields and effectiveness of a particular reactor. Further, the augmentation in algae yield entails better microalgae culture and amplified CO2 capture. In the scale-up process to industrialization, maintenance of controlled conditions in open ponds is the major issue (Fig. 4.2). To maintain the controlled conditions, closed photobioreactors are considered as alternative system against open ponds. Photobioreactor system has several advantages than open pond system except capital and operation costs.

Fig. 4.2
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Schematic diagram of raceway pond for the cultivation of microalgae. (Adapted from Seambiotic 2009)

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Capital cost may be required for the manufacturing, installation, and supporting accessory systems. The operational cost due to the CO2 addition, O2 removal, maintenance and cleaning, etc. will increase the costs. The harvesting cost is low when compared with the open ponds, which compensates the initial costs. The other important factors, i.e., biomass quality, biomass concentration, production flexibility, process controls, and reproducibility are more preferred in the PBRs than the open ponds. In case of open ponds, the requirement is huge space, water, and CO2 loss and the contamination risk is also more.

4.6.1 Closed Reactor Designing for Microalgae Cultivation

Generally, the reactor is in cylindrical shape and fits with several accessories for proving specific temperature, pH, and mixing devices. Algae cultivation needs light source as energy which can be provided by a light source or sunlight for photosynthesis. Since algae cultivation is through a light source, these reactors are calling as photobioreactors (PBR). PBRs offer a protection (Fig. 4.3) from the contaminated species (bacteria, fungi, etc.) and make available closed environments to maintain desirable temperature, pH, mixing, etc. and ensure the specific algal strain cultivation. The below-mentioned variables are essential features that must be considered in the design of closed reactor system: (1) light source and orientation, (2) nutrition by culture medium, (3) water, (4) algae circulation (mixing), (5) pH, (6) temperature control, (7) CO2 feed, (8) O2 removal, (9) materials of construction, and (10) reactor maintenance. The parameters remains the same but there may be changes in their intensities as per the array of higher and lesser points for specific strain cultivation management.

Fig. 4.3
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Tubular photobioreactor (a), open pond (b) for microalgae cultivation. (Adapted from Li et al. 2008)

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4.6.2 Classification of Photobioreactors (PBRs)

Photobioreactors are classified into various types depending upon the design of reactors, i.e., (a) tubular or flat, (b) horizontal, (c) inclined, (d) vertical or spiral, (e) manifold or serpentine, (f) hybrid, (g) floating, and (h) biofilm reactors.

Depending on the mode of operation, photobioreactors are categorized as (1) single-phase reactors and (2) air-mixed reactors. In single-phase reactors, gas exchange takes place, whereas in air-mixed reactors, both liquid and gas mass transfer occurs.

Based on the construction materials, again PBRs are classified into (a) glass or plastic and (b) rigid or flexible photobioreactors. Other than these, axenic PBRs are used for specific cultures and intended for sterilization before the operation of reactor.

4.6.2.1 Light

Light is an essential energy for photosynthesis; light must be radiated inside the reactor to avoid the dark zones (less-intensity light at some places in the reactor) or illuminate the fluorescent lights close to the outer surface of the reactor. When the light penetrates with high intensity and reaches to the cells with good frequency in a reactor then it is considered as an optimal reactor. Spatial dilution of illumination is a significant feature which reduces the mutual shading of cells in the culture medium. This may lead to higher growth rate, but the product component inside the cell is less. One can consider how to exploit the volume to surface area ratio and geometry of reactor for efficient light distribution in the designing process of PBR (Fig. 4.4).

Fig. 4.4
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The typical photobioreactor for microalgae production

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The light gradient directly influences the productivity of cultured algae and is determined by the reactor dimensions and algal cell density. The cell density formation is a microalgae strain specific property, and it is essential to manage optimal conditions for the light penetration and light intensity to reach each and every cell in the reactor. The critical cell density is an operating parameter, which is nothing but the greatest cell mass without reciprocated shadows and one can take into consideration this point while designing the PBR. Naturally light/dark periods also influence the efficacy of photosynthesis of the microalgae. Janssen et al. (2001) explained the impact of illumination on photosynthetic effectiveness of D. tertiolecta and on short irradiance or dimness compared with continuous light. Photon flux of the illumination tenure and time of liquid duration in various radiance regions are two key parameters to attain the optimal dark period. The light regime of PBR is influenced by the extent of light/dark periods and illumination strength, and the light regime directly responsible for physiological responses of cells is called as photoacclimation. Zu and Richmond (2000) reported that the sudden flash of light is fatal for various algal species. Based on the photosynthetic efficiency and other data, the theoretical maximum fuel yield per area can be determined. LEDs are more prominently used as light source in algal cultivation, due to low cost, and can also use it as flash light to pulse the light whenever it is required to improve the productivity.

4.6.2.2 Mixing

Mixing is the critical parameter which enhances the rate of recurrence of the illumination and murk cycles and leads to improve the productivity and cellular contents. It provides the effective mass transfer from nutrients to the cell in the presence of light. In the designing of the bioreactor, effective mixing will be considered for effective availability of nutrients and light to the cell by avoiding the diffusion barriers. Ugwu et al. (2008) scale out tubular reactors by setting up of static mixers with augmented tube diameter and confirmed that the enhanced light exploitation and microalgae mass improved yields. For optimal cell growth, the reactor maintains the capability to sustain the mass transfer coefficient (kLa), it build upon the category of bubbler, agitation rate, type of surfactants and also on temperature. Static mixers installation could reduce the bubble size which increases the surface area and leads to increase the contact area in between liquid and gas. Whereas fine spargers produce low liquid flow rate, and big bubble formation leads to low or poor mass transfer. Eriksen et al. (1998) explained a setting up of dual orifice with membrane-perforated sparger in a closed reactor, which resulted in efficient air and CO2 supply separately and attained proper mixing. They achieved fivefolds supply of CO2 to liquid phase as compared to conventional spargers. In the total cost of the reactor, usually the highest percentage is occupied by energy consumed and the mixing mechanism. In bubble column reactors, the sparger working efficiently mixing and formed small size bubbles thereby efficient CO2 mass transfer takes place.

The specialty of this type of reactor with no moving parts is that it provides elevated diffusion area volume ratio, excellent mass and heat transmittance, excellent release of O2, and also small capital cost. This was scaled up by perforated plates that are set inside the reactor, resulting in break-up of coalesced particles and are redistributed. An airlift reactor is also one type of bubble column reactors; it has internal draft tube which enhances the mixing and mass transfer coefficient. In Fig. 4.5, the left side designated three zones: air-riser up, bubble down-comer, and disentanglement zones in air lift reactor.

Fig. 4.5
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Description of the bubble column PBR (a) and airlift PBR (b)

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Air bubbles are sprinkled by sparger through the bottom of the reactor, maybe inside or outside of the draft tube. The air bubbles go up (rise) and lead to fluid flow vertically, due to the opposite action against this, and the fluid flow will be downwards causing down flow. Upon the continuous supply of air bubbles, there is an effective movement that leads to improved mixing. Further, this movement facilitates in uniform distribution in one direction with same velocity, thereby no or less coalescence, and no fusion of bubbles ensuing greater kLa (the volumetric mass transfer coefficient) than bubble-column reactors. The above positive parameters result in greater mass yields in airlift reactors than other stirred reactors. Several researchers are examined on improved mixing and mass transfer in flat-panel reactors (Wondraczek et al. 2013; Hoekman et al. 2012; Posten and Schaub 2009; Tredici and Materassi 1992).

4.6.2.3 Water Consumption

Water utilization is an important parameter as it is precious along with reactor options. Algae are able to grow in various water sources, i.e., fresh drinking water, brackish water (saline), and also in waste water (Wogan et al. 2008) effluents. Based upon the type of water (pure or saline), strains of microalgae are classically divided into two categories: freshwater algae and salt water algae. The overall productivity and individual content (lipids, carbohydrates, etc.) productivity in each strain are influenced by the level of salinity. Abu-Rezq et al. (1999) reported on the saline conditions of high-quality marine algae for the production, i.e., Nannochloropsis salinity range was 20–40, Tetraselmis 20–35, and Isochrysis 25–35 (Rao et al. 2007).

Waste water having rich nutrients (nitrogen and phosphorus) with high concentrations may be treated further when used as medium for algae cultivation. In open ponds, due to evaporation of water, high concentration of nutrients will be formed; high concentrations of these nutrients causing for growing of unwanted algae and may be undergone eutrification. If it occurs in the salt water ponds, where the salinity of the pond reaches the extreme and above-tolerance levels. In PBRs, by minimizing the evaporation/loss of water and minimizing these extreme concentrations of nutrients in waste water, the unwanted algae growth can be avoided, and these nutrients can be utilize efficiently for the particular strain of algae growth (Aslan and Kapdan 2006). One of the huge costs-saving approaches in algae cultivation is usage of waste water as medium. Chinnasamy et al. (2010) used corporate industry waste water as medium for the cultivation of algae in vertical tank reactors, polybags, and also raceway ponds for mass cultivation of algal consortium. They attained huge proteins and lesser carbohydrates in algal consortium. Yang (2011) reported the same as above results that the overall biomass productivity in poybags is more than the vertical tank reactor and raceway. Attractively, with diverse positioning, the polybag reactor can attain the highest yields in commercial systems (large scale) and diminish running costs.

4.6.2.4 CO2 Consumption

Carbon is essentially required for photosynthesis, i.e., organic and inorganic carbon (CO2), among them inorganic carbon is the best in commercial aspect. For photosynthesis of algae, sufficient CO2 is essential along with light, nutrients, water, and high partial pressures (pCO2). Several studies revealed that excess partial pressure of CO2 is detrimental for microalgae. Lee and Tay (1991) reported that exposure of CO2 with high partial pressures on Chlorella pyrenoidosa leads to declining the rate of growth. They suggested that the supply of CO2 is through a gas permeable membrane to provide controlled release for the entire culture and prevent high partial pressures of CO2. Even though the laboratories routinely aerate 5–15% of CO2, the available concentration from 1 to 5% is often enough for maximum growth (Suh and Lee 2003). Flue gas is the best for carbon source as it reduces the greenhouse gas emissions and also low cost. Thermal power stations are good sources for exhaust gas which exhibit up to 13% of carbon dioxide, and it is of lower cost compared to pure CO2 supply. In the photobioreactor, infrared analyzer is used to measure the CO2 concentration in the gas phase through which the flow of flue gas is regulated. Schenk et al. (2008) reported that pCO2 is 0.15 kPa is the minimum requirement to avoid the limitation for CO2 kinetics, and the required CO2/g dry biomass is 1.7–1.8 g (Chisti 2007). Doucha et al. (2005) explained that even flue gas contains CO and NOx along with CO2; however they may not interfere in the photosynthetic efficiencies even with low mass transfer of CO2, and interestingly the productivity was similar to pure CO2 supply. For further decreasing the cost of CO2 in biofuel production by microalgae, Carvalho et al. (2006) suggested an option that is the use of hollow fiber membranes for interrupted supply of CO2 and enhanced mass transfer.

4.6.2.5 O2 Removal

In algal photosynthesis, oxygen is delivered as byproduct, the maximum allowable is as 10 g O2 m3 min−1 in a typical tubular reactor. Likewise, in a photobioreactor, the dissolved oxygen when excess than the minimum allowable levels leads to photooxidative damage in the presence of intensive irradiation ultimately causing to the drastically reduced productivity. In general, O2 concentration should be 400% of air saturation. In case of open ponds, oxygen is evolved from the medium easily, but in photobioreactors, the oxygen pressure is built inside, so periodically oxygen should be pumped out of the reactor. In tubular reactors, it is very difficult to do it, which limits the scale-up process. Molina et al. (2011) designed the airlift zone by algae culture frequently returned in the tubular reactor where oxygen was stripped by air. In the airlift zone, gas–liquid separator prevents recirculation air bubbles to horizontal loop. The fluid travel time through the extent of the degasser should be the same as oxygen bubbling rising time.

4.6.2.6 Nutrient Supply

Nitrogen and phosphorus are the main nourishments to manage the growth rate and yield of lipid in algae. The essential other nutrients required for microalgae are carbon, oxygen, hydrogen, sulfur, chlorine, sodium, calcium, potassium, and magnesium. The micronutrients are iron, silicon, cobalt, nickel, copper, manganese, molybdenum, boron, vanadium, and selenium. In N-8 medium (common medium) containing deficient quantities of Mg, S, Fe and N which results huge cell mass. Even in separate supplementation of these elements not showed better yields, but the four elements reasonable boosting reported enhanced performance. Chisti (2007) explained the need for the excess nutrients like phosphorus which reacts with metal ions. Applying stress condition by providing limited nutrients can enhance the lipid percentages with biomass. But the stress application may limit the rate of growth and decrease the overall lipid production. Rodolfi et al. (2009) illustrated three types of nutrient supply: (1) nutrient-sufficient, (2) nutrient-limited, and (3) nutrient-deficient. They applied the above three condition on few strains, among them Nannochloropsis showed exceptional results, lipid production was enhanced, when applied deficient scenario (N-deficient environment) after grown formerly with nutrient sufficient medium. Xinxin et al. (2019) studied the consequences of N-limitation on fatty acids and lipid quantity on P. tricornutum, Isochrysis galbana, Rhodomonas baltica, and N. oceanica, and higher yields are achieved. Various media are tabulated and their compositions with quantities are discussed in detailed in the cultivation section.

4.6.2.7 Temperature

Temperature is one of the critical parameters which play a vital role for large-scale culturing of microalgae. Because the daily variations may be seen upon the presence of illumination and at dark cycles, which leads to the reduction in biomass production and algal lipid capacity. Microalgae showed decline in cell volume with an enhanced temperature (upon extra illumination), and the maximum growth occurred at the temperature range was noted as 20–30 °C. Venkata Subhash et al. (2014) revealed that several algae species tolerate the temperatures up till 15 °C lesser than their optimistic point, with lower growing rate. But the temperature is fewer degrees more than the tolerable point causing to death of microalgae as it is an environmental parameter (Huang et al. 2010).

Zhu et al. (2013) critically explained that temperature choice may vary from species to species. For example, Scenedesmus and Chlorella were adapted in the temperature range of 5–35 °C; the best range of temperature is 25–30 °C. Kurpan et al. (2015) experimented on Isochrysis galbana, N. oceanica, and P. tricornutum for biodiesel and PUFAs. Isochrysis galbana and Phaecodactylum tricornutum yielded high TAG at 20 °C and 30 °C, respectively, and Nannochloropsis oceanic yield was noted as very less. They stated that, if an ideal temperature was not maintained during the course of cultivation, the biochemical pathways are changed, which leads to improper build-up of lipids. Singh and Singh (2015) reported an optimum temperature of 20–30 °C for the algae, i.e. Nannochloropsis, Chlorella, Scenedesmus, Botryococcus, Spirogyra, Neochloris, Chlamydomonas, Haematococcus, and Ulva species. The studies in the literature report that the optimum growth temperature depends on algal species and geographical origin, where an optimum growth can be attained (Goldman and Carpenter 1974). When the tolerable temperatures exceeds, the response of microalgae against temperature variation could affect (1) nutritional necessities, (2) nature of metabolism and rate, and (3) cell composition (Richmond 1999). Torzillo et al. (1991b) examined the temperature consequences on S. platensis at outdoor environments at different temperatures, i.e., 25 and 35 °C (from May to September). They reported that there is 14% increase in biomass productivity at 35 °C, (1) when the temperature decreases in the period and the average biomass productivity is decreased, and (2) during night, there is a loss of biomass significantly at 25 °C (Torzillo et al. 1991a).

According to geographical regions, microalgae cultivation in outdoors (open ponds and race ponds) may be exposed to extreme temperatures. Even though microalgae competent grows at various temperatures, favorable growth is restricted with a slight range of temperature in particular to each strain. Abu-Rezq et al. (1999) reported the best range of temperatures for Isochrysis, Nannochloropsis, and Tetraselmis were 24–26 °C, 19–21 °C, and 19–21 °C, respectively. Daily and seasonal temperature fluctuations also obstruct the algae growth. In the absence of controlling units in PBRs can result high temperatures, then evaporate cooling method is frequently adapted to reduce that much magnitude from required temperatures. Further at low temperatures especially at night times owing respiration could loss the biomass. So, maintenance of temperature is required for attaining optimum growth in large scale PBRs and in open ponds.

4.6.2.8 pH

pH is an important parameter that influences the microalgae metabolism in lipid production. It influences the fluid chemistry like accessibility of nourishments, organic acids and CO2. Further in marine environment react with water forms (H2CO3) carbonic acid, readily dissociate into bicarbonate (HCO3−) and again detached into carbonate ion (CO3−) and protons (H+) leads to decrease pH ultimately damage the algae growth (Bautista-Chamizo et al. 2018). Bicarbonate (HCO3−) is utilized by microalgae through active transport leads to catalytic conversion of cation exchange and formed as CO2 and OH− (Seyed et al. 2018). Bicarbonate (HCO3−) is utilized by microalgae through active transport, catalytic conversion of cation exchange forms of CO2 and OH− (Seyed et al. 2018). Each and every algal strain has a lesser pH array, so commercial pH regulators must be preferred in reactors to attain the maximum growth. Xinxin et al. (2019) studied the consequences of N-limitation on profile of fatty acids and lipid quantity on P. tricornutum, Isochrysis galbana, Rhodomonas baltica, and N. oceanica and maintained the optimal pH as 8.5. Sharma et al. (2018) studied on Lyngbya confervoides, Chroococcus turgidus, Nostoc commune, Chlorella sp., Skeletonema costatum, and Chaetoceros calcitrans and reported that the suitable pH is the neutral pH. They also stated that the best pH for N. oculata and Chlorella sorokiniana was noted as 8.5 and 8, respectively. For lipid production by Scenedesmus obliquus, it is noted as neutral pH (Breuer et al. (2013), for chlorella sorokiniana optimum pH was 6 (Qiu et al. 2017). Liao et al. (2018) stated that lower pH leads to decreased growth rate, and this effect is reversible and at high intensity. Ramanna et al. (2017) explained about pH as it is specific to strain, set the pH prior to illumination flux to keep away from photo-inhibition which causes irreversible damage to essential proteins.

4.6.3 Other Considerations

When designing a photobioreactor configuration, it is essential to judge the whole process of the production (Table 4.10). Our aim is the highly dense biomass production, in the biomass lipid content and oil downstream processing with good quality. The downstream processing and reactor design integration are very important that impact on the product excellence and cost. Chisti (2008) recommended that the genetic engineering may improve microalgae biomass yield and lipid quantity in cellular level and may have the supreme enhancement in the economics of biofuels. Genetic engineering could improve fuel production in different ways, i.e., upgrading in photosynthesis, enhanced biomass yield, augmented lipid quantity, and better temperature tolerance of algae. In addition, genetic engineering could increase tolerable levels of algal cells to light saturation, photo-inhibition, and photo-oxidation. Geography is important for area selection, assessing the viability of biofuel production, as few regions of the globe are best than other places. In USA, southwest locations are good; Texas is well suitable for large-scale production.

Table 4.10 Various reactor configurations for microalgae with biomass productivity
Full size table

4.7 Conclusions

This chapter discusses the various essential elements required for the cultivation of the microalgae. The basics of the microalgae classification, components present, and cultivation requirements have been explained. A review on the components (from carbohydrates to bioactive compounds) along with the compositions, cultivation, media, and various media used for the cultivation of microalgae with metabolic pathways are consolidated in the tables and figures. In this chapter, around 55 media compositions are tabulated and their specialties and applications for the microalgae as feedstock are explained.