One of the decisions to be taken in the cultivation of microalgae is regarding the use of open or closed photobioreactors. Closed photobioreactors of the vertical tubular, helical tubular, and flat panel type are considered to have high photosynthetic efficiency and degree of control. Closed reactors have some advantages and disadvantages over open ones.

1.1.1 Closed Photobioreactors

Due to the high productivity achieved in cultures carried out in closed photobioreactors, much attention has been paid to these systems. The configurations tested on a laboratory or pilot scale include vertical reactors, flat plate, annular, plastic bags, green wall panel (GWP), and various forms of tubular reactors, stirred mechanically or by airlifting.

Closed photobioreactors are highly efficient at biofixation of CO2, mainly due to better homogeneity of the medium and mass transfer. However, these reactors are limited by the excess O2 produced (Ho et al., 2011). The costs of these reactors are generally high (Table 1.1). Contamination can be controlled in sterile systems; however, this causes an in­crease in production costs (Amaro et al., 2011). The scale-up of open photobioreactors gener­ally occurs by increasing the diameter of the tube, but the cells do not receive sufficient light for growth (Ugwu et al., 2008).

Microalgae are a very heterogeneous group of microorganisms. The term microalgae includes prokaryotes and eukaryotes. Cyanobacteria (blue-green algae) are frequently unicel­lular, with some species forming filaments or aggregates. The internal organization of a cyanobacterial cell is prokaryotic, where a central region (nucleoplasm) is rich in DNA and a peripheral region (chromoplast) contains photosynthetic membranes. The sheets of the photosynthetic membranes are usually arranged in parallel, close to the cell surface. Eukary­otic autotrophic microorganisms are usually divided according to their light-harvesting pho­tosynthetic pigments: Rhodophyta (red algae), Chrysophyceae (golden algae), Phaeophyceae (brown algae), and Chlorophyta (green algae). Their photosynthetic apparatus are organized in special organelles, the chloroplasts, which contain alternating layers of lipoprotein mem­branes (thylakoids) and aqueous phases (Staehelin, 1986).

All photosynthetic organisms contain organic pigments for harvesting light energy. There are three major classes of pigments: chlorophylls (Chl), carotenoids, and phycobilins. The chlorophylls (green pigments) and carotenoids (yellow or orange pigments) are lipophilic and associated in ChI-protein complexes, while phycobilins are hydrophilic. Chlorophyll molecules consist of a tetrapyrrole ring (polar head, chromophore) containing a central magnesium atom and a long-chain terpenoid alcohol. Structurally, the various types of Chl molecules, designated a, b, c, and d, differ in their side-group substituent on the tetrapyr­role ring. All Chl have two major absorption bands: blue or blue-green (450-475 nm) and red (630-675 nm) (Niklas Engstrom, 2012). Chl a is present in all oxygenic photoautotrophs.

Photoautotrophic cultures seldom reach very high cell densities; they are more than an order of magnitude less productive than many heterotrophic microbial cultures, the reason that microalgal cultures are carried in very large volumes. However, the microalgal photosyn­thetic mechanism is simpler than that of higher plants, providing more efficient solar energy conversion. This makes microalgae the most important carbon-fixative group and oxygen producer on the planet. Microalgae cultures have some advantages over vascular plants (Benemann and Oswald, 1996): All physiological functions are carried out in a single cell, they do not differentiate into specialized cells, and they multiply much faster.

Biotechnology is a major interdisciplinary science, combining biology, chemistry, and engineering and incorporating and integrating knowledge from the areas of microbiology, genetics, chemistry, biochemistry, and biochemical engineering. The key word in this context

is biotransformation.

The application of biotechnology to marine organisms and processes is an area of signif­icant industrial importance with ramifications in many areas, including human health, the environment, energy, food, chemicals, materials, and bioindicators. Some areas of interest re­lated to marine biotechnology include the understanding of genetic, nutritional, and environ­mental factors that control the production of primary and secondary metabolites, based on new or optimized products. Furthermore, there has been an emphasis on the identification of bioactive compounds and their mechanisms of action for application in the medical and chemical industry; there are also bioremediation strategies for application in damaged areas and the development of bioprocesses for sustainable industrial technologies (Zaborsky, 1999).

The cultivation of microalgae as part of biotechnology has received researcher attention. The growth conditions and the bioreactors for cultivation have been thoroughly studied (Borowitzka, 1999). The principle behind cultivation of microalgae for the production of bio­mass is the use of photosynthesis (Vonshak, 1997), which involves using solar energy and converting it into chemical energy.

Microalgae are photosynthetic prokaryotic or eukaryotic microorganisms that grow rapidly and have the ability to live in different environments due to their unicellular or simple multicellular structure. Examples of prokaryotic microalgae are the cyanobacteria; green al­gae and diatoms are examples of eukaryotics (Mata et al., 2010).

Cyanobacteria differentiate into vegetative, akinete, and heterocyst cells. The functions of vegetative, akinete, and heterocyst cells are their ability to carry oxygen in photosynthesis, resistance to climactic conditions, and potential for nitrogen fixation, respectively. Green algae have a defined nucleus, cell wall, chloroplasts containing chlorophyll and other pig­ments, pirenoide, and a dense region containing starch granules, stigma, and scourge.

Microalgae exist in various ecosystems, both aquatic and terrestrial. More than 50,000 species are known and about 30,000 are studied (Mata et al., 2010). The main advantages of microalgae cultivation as a biomass source are (Vonshak, 1997):

• They are biological systems with high capacity to capture sunlight to produce organic

compounds via photosynthesis.

• When subjected to physical and chemical stress, they are induced to produce high concentrations of specific compounds, such as proteins, lipids, carbohydrates, polymers, and pigments.

• They have a simple cellular division cycle without a sexual type stage, enabling them to complete their development cycle in a few hours. This enables more rapid development in production processes compared with other organisms.

• They develop in various environmental conditions of water, temperature, salinity, and light.

Lipid accumulation in microalgae usually occurred when microalgae are cultivated under stress conditions (e. g., nitrogen starvation, nutrient deficiency, pH variations, etc.). Among those stress conditions, nitrogen limitation is the most effective and commonly used strategy for stimulating lipid accumulation in microalgae. Recent reports demonstrated that cultivation under nitrogen starvation conditions leads to a marked increase in the oil/lipid content (Mandal and Mallick, 2009). Hu et al. (2008) collected the data of lipid contents of various microalgae and cyanobacteria species under normal growth and stress conditions in a literature
survey, indicating that under stress conditions, the lipid contents of green microalgae, diatoms, and some other microalgae species are 10-20% higher than under normal conditions. However, the lipid contents of cyanobacteria were usually very low (10%) (Hu et al., 2008).

It is thought that when microalgae are cultivated under nitrogen-starvation conditions, the proteins in microalgae will be decomposed and converted to energy-rich products, such as lipids. Siaut et al. (2011) also concluded that during microalgae growth, starch would first be synthesized to reserve energy, then lipid would be produced as a long-term storage mech­anism in case of prolonged environmental stress (such as nitrogen deficiency). Although a nitrogen-starvation strategy is very effective in increasing lipid content of microalgae, the ni­trogen deficiency conditions often lead to a significant decrease in the microalgae growth rate, thereby causing negative effects on lipid productivity. Therefore, engineering approaches should be conducted to optimize the cultivation time for the microalgae growth period (nitrogen-sufficient condition) and lipid accumulation period (nitrogen-deficient condition) to ensure high overall oil/lipid productivity.

The pH values of cultures affect the biochemical processes associated with microalgae, including the bioavailability of CO2 for photosynthesis and use of the medium nutrients.

The optimum pH is determined according to the type of microorganism. Some species have an optimum pH of around 7.0; however, some microalgae are tolerant to high pH (Spirulina, pH 11.0) or low pH (Chlorococcum, pH 4.0) (Kumar et al., 2010).

The optimum growth of the microorganism in an acidic or basic environment can be maintained if the intracellular pH is 7.5, regardless of the external pH. Living cells have the ability, within certain limits, to maintain internal pH by expelling hydrogen ions. The external pH generally has a drastic change before it affects the cell. The optimum pH of the cultures should be maintained, thereby preventing the collapse of cell cultures by the cellular process of rupture due to high pH. The control of pH must be integrated with the aeration system by the addition of alkaline solution to the culture (Wang et al., 2012).

Some microalgae have high productivity when maintained at an alkaline pH between 10 and 11. The high pH may be beneficial for outdoor cultivation because it inactivates patho­genic microorganisms and other microalgae (Kumar et al., 2010).

In the case of cultivation with addition of CO2, the concentration of this gas may be the dominant factor that will determine the pH of the culture. In this case, the CO2 demand results from the balance between the transfer of CO2 to the liquid and CO2 consumption by the cells (Wang et al., 2012). SOx and NOx, present in flue gas from burning coal, can also cause changes in pH, damaging microalgal cultivation. With high concentrations of CO2 the pH drops to 5.0, and when exposed to SOx and NOx this value is 2.6 (Westerhoff et al., 2010).

The pH also influences the removal of ammonia and phosphorus. The high pH may increase the removal of ammonia through its volatilization and phosphorus through its pre­cipitation (Craggs, 2005).

Tubular systems are widely used as close systems in commercial production. Usually tubu­lar photobioreactors are made of transparent polypropylene acrylic or polyvinylchloride

pipes with small internal diameters to increase the penetration of light. Mixing and agitation of the culture are maintained by an air pump to provide circulation (see Figure 2.4).

The most significant characteristic of this tubular system that is different from the vertical column bioreactor is the improvement of air-residence time inside the tubular bioreactor, which can provide more dissolved CO2. These systems could use artificial light, but they are also designed based on natural light (sunlight) provided from outside of the tube. The hydrodynamic stress on the algae may vary, depending on the flow characteristics of each system (e. g., turbulent flow, pump type). Likewise, the gas transfer to the culture may vary from low to high, depending on the flow characteristics and the air-supply technique adopted. The operational difficulties are similar to other systems, including growth of microalgae on the wall of the tubes, thus blocking the light penetration; high oxygen concen­tration that can inhibit photosynthesis; and limits on the length of the tube in a single run (Briassoulis et al., 2010).

Coil-type systems are often adopted to enhance the efficiency of space utilization com­pared to the other categories. Among the most important advantages of the system and com­mon to most coil-type systems are the larger ratio of surface area to culture volume to receive illumination effectively, as well as the easy control of temperature and contaminants (Briassoulis et al., 2010). The cleaning problems of tubular systems are not easy to overcome due to the small internal tube size, which has no ready mechanical way to conduct the inside cleaning for a long tube. The scale-up of these systems is relatively easy compared with other photobioreactor designs. The increase of tubular photobioreactor working volume can easily be achieved by simply extending the tube length to the designed volume if the air pump can affordably provide enough power to pump in air bubbles.

Light

source

Microalgae tank

і

Gas mixer

CO2 tank

FIGURE 2.4 Horizontal tubular photobioreactors for microalgae cultivation.

Hybrid cultivation is a method that combines different growth stages in two types of photobioreactors, closed and open (Brennan and Owende, 2010). The hybrid culture system is designed to utilize the good qualities of both types of reactors. In the case of open and closed reactors, the first stage of the cultivation occurs in the closed photobioreactor, where the con­ditions are controlled to minimize contamination of other microorganisms and to promote continuous cell division.

In the second stage of production the cells are exposed to a nutritional stress, increasing the synthesis of a specific metabolite, such as lipids, proteins or polymers. The second stage is ideal for open ponds (Brennan and Owende, 2010). The a-shaped reactor is another type of hybrid system, developed by Lee et al. (1995). In this reactor, the culture is lifted 5 meters by air to a receiver tank, and culture flows down an inclined PVC tube (at an angle of 25° to the horizontal) to reach another set of air-riser tubes, and the process is repeated for the next set of tubes.

Photosynthesis can be defined as a redox reaction driven by light energy, in which carbon dioxide and water are converted into metabolits and oxygen. Photosynthesis is traditionally divided into two stages, the so-called light reactions and the dark reactions. The first process is the light-dependent process (light reaction), which occurs in the grana and requires the direct energy of light to make energy carrier molecules that are used in the second process. The light-independent process (or dark reaction) occurs in the stroma of the chloroplasts, where the products accumulated in the products of the light reaction are used to form C-C covalent bonds of carbohydrates. The dark reactions can usually occur if the energy carriers from the light process are present.

In the light reactions, light strikes chlorophyll a in such a way as to excite electrons to a higher energy state. In a series of reactions, the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a byproduct of the reaction. The ATP and NADPH are used to make C-C bonds in the dark reactions.

In the dark reactions, carbon dioxide from the atmosphere (or water for aquatic and marine organisms) is captured and reduced by the addition of hydrogen to form carbohydrates ([CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbon fixation. The energy comes from the first phase of the photosynthetic process. Living systems
cannot directly utilize light energy, but they can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes. So, the main role of the light reactions is to provide the biochemical reducing agent NADPH2 and the chemical energy carrier (ATP) for the assimilation of inorganic carbon, as presented in Equation 4.1:

2NADP + 3H2O + 2ADP + 2Pi $ 2NADPH2 + 3ATP + O2 (4.1)

The fixation of carbon dioxide happens in the dark (in the stroma of chloroplasts) using the NADPH2 and ATP produced in the light reaction of photosynthesis (Equation 4.2):

CO2 + 4H + 2NADPH + 3ATP $ (CH2O) (4.2)

Carbon dioxide is available in water in three different forms: CO2, bicarbonate (HCOg), or carbonate (HCO32~) (Figure 4.1), the relative amounts of which are pH dependent. Although plants and algae are known to be dependent exclusively on the Calvin-Benson-Bassham cycle (also known as the Calvin cycle) (Atomi, 2002), six autotrophic carbon-fixation pathways are known. These are (1) the Calvin cycle, (2) the acetyl-CoA pathway, (3) the 3-hydroxypropionate cycle, (4) the reverse tricarboxylic acid cycle, (5) 3-Hydroxypropionate/4-hydroxybutyrate cycle, and (6) Dicarboxylate/4-hydroxybutyrate cycle (GeorgeFuchs, 2011). This section discusses the Calvin cycle, which is the most important in microalgae.

In the Calvin cycle there is only one enzyme responsible for CO2 fixation: ribulose 1,5-biphosphate carboxylase/oxygenase, also known as Rubisco. Figure 4.2 shows the Calvin

CO2(g) ^—► CO2(aq) ^—► H2C03 ч—► HCO — ч—► C032- FIGURE 4.1 Different forms in which carbon dioxide is

available in water.

cycle, where one molecule of ribulose 1,5-biphosphate and a CO2 are converted into two glycerate phosphate. CO2 diffuses through the cell and is captured by the enzyme ribulose biphosphate (Rubisco).

CO2(g) $ CO2 (aq) $ H2CO3 $ HCO3 $ CO32- The fixation of CO2 occurs in four distinct phases (Masojidek et al., 2004):

1. Carboxylation. A reaction whereby CO2 is added to the five carbon sugar ribulose bisphosphate (Ribulose-bis-P) to form two molecules of phosphoglycerate (Glycerate-P). This reaction is catalyzed by the enzyme ribulose biphosphate carboxylase/oxygenase (Rubisco).

2. Reduction. To convert Glycerate-P into 3-carbon sugars (Triose-P), energy must be added in the form of ATP and NADPH2 in two steps, which are the phosphorylation of Glycerate-P to form diphosphoglycerate (Glycerate-bis-P) and the reduction of Glycerate-bis-P to phosphoglyceraldehyde (Glyceraldehyde-P) by NADPH2.

3. Regeneration. Ribulose-P is regenerated for further CO2 fixation in a complex series of reactions combining 3-, 4-, 5-, 6-, and 7-carbon sugar phosphates, which are not explicitly shown in the diagram.

4. Production. The primary end products of photosynthesis are considered to be carbohydrates, fatty acids, amino acids, and organic acids.

Besides the carboxylase activity described here, all Rubiscos (there is more than one type) are known to display an additional oxygenase activity in which an oxygen molecule, com­peting with CO2 for the enzyme-bound eno-diolate of RuBP, reacts with RuBP to form

3- phosphoglycerate and phosphoglycolate (Atomi, 2002). The latter product is subse­quently oxidatively metabolized via photorespiration, leading to a net loss in carbon dioxide fixation. Photorespiration thus represents a competing process to carbon fixation, where the organic carbon is converted into CO2 without any metabolic gain. Photorespiration depends on the relative concentrations of oxygen and CO2 where a high O2/CO2 ratio stimulates this process, whereas a low O2/CO2 ratio favors carboxylation. Rubisco has low affinity by CO2; its Km (half saturation) is approximately equal to the level of CO2 in air. Thus, under high irradiance, high oxygen level, and reduced CO2, the reaction equilibrium is shifted toward photorespiration. For optimal yields in microalgal mass cultures, it is necessary to minimize the effects of photorespiration, achieved by an effective stripping of oxygen and by CO2 enrichment. For this reason, microalgal mass cultures are typically grown at a much higher CO2/O2 ratio than that found in air, which is in turn an opportunity to reuse industrial gas emissions.

The source of nitrogen in cultivation of microalgae seems to cause changes in oxygen production during photosynthesis. The ratio between O2 evolution rate and CO2 uptake rate (the photosynthetic quotient, PQ) depends on the composition of the produced biomass and the substrates that are used. Especially oxidized nitrogen sources, which must be reduced before they are incorporated into the biomass, affect the PQ. When nitrate is used, it is expected at an evolution of 1.3 mol O2 per mol of CO2 assimilated, whereas nitrite promotes a release of 1.2 mol O2 and ammonia 1.0 mol O2 (Eriksen et al., 2007). Approximately 20% of O2 evolution equivalents can be accounted for by NO33 uptake and assimilation under N-replete conditions (Turpin, 1991).

Under phototrophic growth conditions, microalgae absorb solar energy and assimilate car­bon dioxide from the air and nutrients from aquatic habitats. However, commercial produc­tion must replicate and optimize the ideal conditions of natural growth. The choice of the reactor is one of the main factors that influence the productivity of microalgal biomass.

Open tanks come in different forms, such as raceway, shallow big, or circular (Masojidek and Torzillo, 2008). Circular ponds with a centrally pivoted rotating agitator are the oldest large-scale algal culture systems and are based on similar ponds used in wastewater treat­ment. The design of these systems limits pond size to about 10,000 m2 because relatively even mixing by the rotating arm is no longer possible in larger ponds. Raceway tanks are the most widely used artificial systems of microalgal cultivation. They are typically constructed of a closed loop and have oval-shaped recirculation channels. They are usually between 0.2 and 0.5 m deep, and they are stirred with a paddlewheel to ensure the homogenization of culture in order to stabilize the algal growth and productivity. Raceways may be constructed of concrete, glass fiber, or membrane (Brennan and Owende, 2010).

Compared to closed tanks, the raceway is the cheapest method of large-scale microalgal production (Chisti, 2008). These tanks require only low power and are easy to maintain and clean (Ugwu et al., 2008).

The construction of open tanks is low cost and they are easy to operate; however, it is dif­ficult to control contamination, and only highly selective species are not contaminated by other microalgae and microorganisms. Environmental variations have a direct influence, and the maintenance of cell density is low due to shadowing of the cells (Amaro et al.,

2011) . Light intensity, temperature, pH, and dissolved oxygen concentration may limit the growth parameters of open tanks (Harun et al., 2010).

Open photobioreactors have lower yields than closed systems due to loss by evaporation, temperature fluctuations, nutrient limitation, light limitation, and inefficient homogenization (Brennan and Owende, 2010). The amount of evaporated water can be periodically or contin­uously added to the raceway. The amount of evaporated water in raceways depends on the temperature, wind velocity, solar radiation, and pressure of water vapor. Water can also be lost during harvesting; however, recycling of the medium reduces this problem, and nutrients from the culture medium can be reutilized (Handler et al., 2012).

Open ponds are the microalgal cultivation systems that have been studied for the longest time. These reactors are used on an industrial scale by companies such as Sosa Texcoco, Cyanotech, Earthrise Farm, Parry Nutraceuticals, Japan Spirulina, Far East Microalga, Taiwan Chlorella, Microbio Resource, Betatene, and Western Biotechnology (Spoalore et al., 2006). Earthrise Farm began cultivation on a large scale in 1976 with Spirulina. Currently the company produces Spirulina and Spirulina-based products. The cultures are grown in 30 open ponds that are 5,000 square meters in size, each one mixed by a 50-foot paddlewheel (Earthrise, 2012).

Since 1981 Parry nutraceuticals has produced Spirulina in powder form, capsules, pills and tablets, and extracts astaxanthin from Haematococcus pluvialis. The company is located in South India (Oonaiyur), and the crops are grown in open ponds, covering an area of 130 acres (Parry Nutraceuticals, 2012). Cyanotech, located in Kailua Kona, Hawaii, on the Pacific Ocean, develops and markets astaxanthin from Haematococcus in gel capsules and Spirulina in tablet form in an area of 90 acres. Since 1984, Spirulina has been cultivated in open ponds, with the medium supplemented with water from the Pacific Ocean and agitation by paddlewheels (Cyanotech, 2012).

In Brazil, since 1998 the Laboratory of Biochemical Engineering (LEB) at the Federal University of Rio Grande (FURG) has been developing a project that studies the cultivation of Spirulina on a pilot scale in an open pond (Figure 1.1) on the edge of Mangueira Lagoon (Morais et al., 2009), for addition to meals for children. Products that are easy to prepare, con­serve, and distribute have been developed. These products include instant noodles, flan, powdered mixture for cakes, cookies, chocolate powder, instant soup, isotonic sports drinks, gelatin powder, and cereal bars (Costa and Morais, 2011).

The LEB, along with the President Medici Power Plant (UTPM), operated by the Society of Thermal Electricity Generation (CGTEE) since January 2005, has carried out the cultivation of microalgae for the biofixation of CO2 that is emitted in the combustion of coal at UTPM in an open pond (Figure 1.2) (Morais and Costa, 2007).

In southern Brazil, the company Olson Microalgae began commercial production of Spirulina capsules as a nutritional supplement in 2012, with an annual target of 6,000 kg in open ponds (Figure 1.3).

The Company Ouro Fino produces biomass and protein from microalgae for human and animal feed using the culture medium vinasse, cane husks, and carbon dioxide generated from the alcohol industry (Figure 1.4).

Several studies of open systems have taken place. According to Lee (2001), only species with high resistance are grown in open systems, such as Dunaliella (resistant to high salin­ity), Spirulina (grown in high alkalinity), and Chlorella (grown with high nutrient concentrations).

The type of light source is known to be a critical factor affecting the growth of microalgae due mainly to the difference in the coverage of wavelength range (Terry, 1986). In addition to the type of light source, the light intensity is also very important for microalgae growth (Grobbelaar et al., 1996; Sanchez et al., 2008; Ugwu et al., 2008; Yoon et al., 2008). In general, the effect of light intensity on the photoautotrophic growth of microalgae could be classified into several phases, such as the light-limitation phase, the light-saturation phase, and the light- inhibition phase (Ogbonna and Tanaka, 2000). To maximize biomass productivity, the satura­tion light intensity needs to be distributed throughout the entire microalgae cultivation system. However, this is impossible in practical cultivation systems, since the light distribution inside the photobioreactor normally decreases significantly along with the distance due to the light shading effects (see Figure 2.1), especially when the cell concentration gets very high or when

FIGURE 2.1 Effect of light intensity on specific growth rate of microalgae under phototrophic cultivation (Ogbonna and Tanaka, 2000).

significant biofilm formation on the surface of the reactor vessel occurs (Chen et al., 2008). Im­proving the mixing of the cells can reduce the effects of light shading or photoinhibition at dif­ferent zones of the photobioreactor. Some literature describes the effect of light intensity on the lipid content of microalgae. Lv et al. (2010) demonstrated that in comparing low and high light intensity (i. e., light-limitation and light-saturation conditions), using a light intensity of 60 gmol/m2/s led to an increase in biomass concentration and lipid content of Chlorella vulgaris, along with changes in pH, NADPH, and Mg2+ concentration (Lv et al., 2010).