Microalgal cultures are susceptible to contamination by different species of microalgae, bacteria, viruses, fungi, protozoa, and rotifers. The contamination by other microorganisms can cause changes in the cell structure and reduce the concentration and microalgal yield in just a few days (Park et al., 2011). These are controlled in open ponds by effectively operating the system as a batch culture and restarting the culture at regular intervals with fresh water and unialgal inoculum. Other contaminants include insects, leaves, and airborne material. It is essential to control this contaminants within acceptable limits. In open ponds, large con­taminants can be removed regularly by placing a suitably sized screen in the water flow. This can be done manually or it can be automated.

Some characteristics can make cultures more susceptible to contamination, such as cultures in continuous mode. According to the characteristics of the microalgal species used, one can apply techniques to maintain an axenic culture. Some of these techniques are maintaining the process of cultivation at an alkaline pH (9.0 to 11.0), using high concentrations of nutrients or salinity, and using antibiotics. The photobioreactor must be periodically cleaned to minimize the chances of contamination (Wang et al., 2012).

If the microalgal biomass is applied to products such as biofuels, waste treatment, biofertilizers, or biofixation of CO2, impurities are acceptable in the microalgal cultivation. However, for bioproducts such as drugs and food, crops must be kept in axenic cultures (Wang et al., 2012).

Despite the fact that great progress has been made in developing photobioreactors for mass production of microalgal cells, more efforts are still required for further improvement, espe­cially regarding the cost reduction of bioreactor design. For large-scale outdoor microalgae cultivation, large amounts of required land space are still the critical issue. In addition, since outdoor photobioreactors usually utilize natural solar light and without additional temper­ature control, the growth of biomass would greatly depend on weather conditions and am­bient temperature. Due to these limitations, in most regions of the world it is not feasible to have stable microalgal biomass production through outdoor mass cultivation. In addition, the potential contamination is also a serious threat to the operational success of outdoor open ponds or raceways. In contrast, closed system photobioreactors have the advantages of better operational stability and condition control. However, the high equipment cost and process cost of closed photobioreactors are still barriers impeding the mass cultivation of microalgae. Finding more rigid, reliable, and transparent materials with lower costs for the design of closed photobioreactors is crucial to enhance cultivation efficiency and to reduce the cost of photobioreactors for the development of closed systems for the autotrophic cultivation of microalgae.

Microalgae grow in soil unfit for agriculture and livestock and in lakes or ponds located in inhospitable lands, such as deserts, which are usually unsuitable for generating any kind of food. Microalgae can double their biomass in a period of 3.5 days, achieving high yields (Chisti, 2007). After harvesting and drying of the biomass, the final state of the product is a powder. According to the chemical composition of microalgae, the biomass may have several applications.

Studies on microalgae are preferably done under controlled conditions. Microalgae biore­actors are often designed differently from bioreactors used to grow other microorganisms. Two parameters are the most important in algae cultivation: efficiency of light utilization and availability of dissolved CO2.

Like any organism, microalgae have nutritional requirements: carbon sources, energy, water, and inorganic nutrients. In the case of microalgae, the carbon source can be CO2 and the energy comes from sunlight. As microalgae grow in aqueous suspension, the manip­ulation and control of culture conditions makes their cultivation feasible, thus the productiv­ity is limited mostly by the available of light. Responses by algal cells to nutrients and cultivation environments can be used to manipulate the processes to favor the production of algal biomass (Benemann et al., 2002).

The development of media for microalgae cultivation involves a sufficient carbon source (carbon is a part of all the organic molecules in the cell, making up as much as 50% of the algal biomass); salt concentration (depending on the original biotope of the alga); nitrogen (represents about 5-10% of microalgae dry weight); phosphorus (part of DNA, RNA, ATP, cell membrane); sulfur (constituent of amino acids, vitamins, sulfolipids and is involved in protein biosynthesis); potassium (cofactor for several enzymes and involved in protein syn­thesis and osmotic regulation); magnesium (the central atom of the chlorophyll molecule); iron (constituent of cytrochromes and important in nitrogen assimilation); pH of the medium; temperature; trace elements, and addition of organic compounds and growth promoters.

Carbon is important because it is the source of energy for many cellular events (such as metabolites production) and reproduction and is part of the physical structure of the cell. In conditions of low dissolved inorganic carbon (DIC), a DIC transport is induced in most microalgae (Matsuda and Colman, 1995), allowing normal cell growth.

Depending on the material used in cultivation of microalgae and the utilization of biomass, three different systems can be distinguished (Becker, 1994):

1. Systems in which a selected algal strain is grown in a so-called clean process, using fresh water, mineral nutrients, and carbon sources. The algae in such systems are intended to be utilized mainly as food supplements.

2. Systems using sewage or industrial wastewater as the culture medium. The cultivation of the microalgae involves secondary (BOD removal) and tertiary (nutrient removal) treatments and production of biomass-based products.

3. Cultivation of algae in enclosed systems under sunlight or artificial light, with cells preferably being grown in autotrophic media.

Microalgae are microorganisms that are capable of producing many different compounds of industrial interest, some with high and some with low aggregated value. The final value of the product and its destination directly influence the conditions of cultivation. Therapeutical compounds produced by microalgae, for example, must be produced through a totally con­trolled and clean process, whereas for the fuel industry residues can be used and the control of the process can be less accurate. The low culture concentration and the corresponding high downstream costs define production trends.

The utilization of complex media (those of which the composition is not determined, such as industrial residues) in the cultivation of microalgae is one alternative to make the produc­tion of some microalgal metabolites economically feasible. Associated with residue compo­sition and microalgae metabolism, knowledge of the needs of the microalgae might save time (and money) in the development of a process. It is very important to supply all microalgae chemical needs because it is known that variations in the chemical composition of phytoplankton are also tightly coupled to changes in growth rate (Goldman et al., 1979).

The type and design of photobioreactors for large-scale cultivation of microalgae represent a compromise between the cost of investment and establishment of optimal conditions for obtaining maximum productivity. The cultivation of microalgae can be carried out in various types of bioreactors (Vonshak, 1997). The microalgae Spirulina and Chlorella are the most commonly cultivated in open ponds around the world.

When choosing the appropriate cultivation system, many parameters must be observed:

• Biology of the microalga

• The cost of land, energy, water, and nutrients

• Local climactic conditions

• Final product

The ability of microalgae to survive in marine environments has received considerable attention. It was found that microalgae can produce some metabolites to protect salt injury and to balance the influence of osmotic stresses of the surroundings. The microalgae, bacteria, and cyanobacteria can tolerate up to 1.7 M of salt concentration in marine medium. The salinity condition may stimulate the production of specific components in microalgae. For instance, Fazeli and his colleagues reported that the highest carotenoid contents (11.72 mg/L) of Dunaliella tertiolecta DCCBC26 occurred when the culture medium contained

0. 5 M NaCl (Fazeli et al., 2006). However, salinity conditions may cause negative effects on the microalgal growth. It was reported that a salinity of 35% (standard seawater) or higher led to a reduction in the growth rate and the efficiency of photosynthesis and dark respiration (Jacob et al., 1991).

The harvesting of biomass is the removal of biomass from the culture medium. This process can involve one or more steps, including chemical, physical, or biological methods. The tech­niques of recovering microalgal biomass from the culture medium can contribute to 20-30% of the total cost of the biomass (Mata et al., 2010). Some techniques for harvesting the biomass include sedimentation, flocculation, centrifugation, filtration, flotation, and electrophoresis. The costs of these operations are relatively high due to the low initial concentration of biomass and the fact that the cells are negatively charged and due to an excess of organic material, which contributes to its stability in a dispersed state (Brennan and Owende, 2010).

The selection of an appropriate harvesting method depends on the properties of the microalga, the cell density, size, and the desired specifications of the final product. The harvesting of the biomass has two steps: separation of the microalgal biomass from the culture medium and the concentration of biomass with removal of excess medium (Amaro et al., 2011).

Many companies are currently engaged in algae-based biofuel research, but players with large-scale production abilities are still few. According to a recent article (Jacquot, 2009), the leading companies in this field are Algenol Biofuels, Sapphire Energy, Seambiotic, Solazyme, and Solix BioSystems (ordered alphabetically). Mass cultivation to offer algae biomass as starting materials is critical to these algae-based biofuel companies. Based on the information on the Websites of these five leading companies, they all developed their proprietary and spe­cialized cultivation methods (see Table 2.3), including photobioreactor systems, open pond systems, and fermentation systems.

Algenol developed a technology, known as Direct to Ethanol®, to produce ethanol from cyanobacteria. Two central components in this technology are gene-modified cyanobacteria and a flexible plastic-film photobioreactor. The genetically modified cyanobacteria can overexpress fermentation pathway enzymes and enhance the ethanol production (see Figure 2.5). The photobioreactors Agenol uses are constructed of flexible plastic film. Each photobioreactor consists of ports for ethanol collection and the introduction of CO2 and nutrients, a mixing system, and ethanol collection rails (see Figure 2.6). Therefore, Algenol claims that they produce biofuel directly from the algae without killing or harvesting the creatures.

Solix also uses photobioreactors to cultivate algae, and they have named their system the Lumian Algae Growth System (AGS ). The AGS system comprises a network of thin panels held in a shallow water bath. The commercialized AGS system is the Lumian AGS4000, which is a 4,000-liter cultivation system with 20 200-liter Lumian panels held in a 12 x 60-foot water — filled system (see Figure 2.7). Furthermore, this system is integrated with a support system for

Company Founded Name Time Location Biofuel Type Technology Cultivation Equipments Market
Algenol 2006 Biofuels	Algenol’s patented technology (known as Direct to Ethanol® Technology) enables the production of ethanol for less than $1.00 per gallon and targets commercial production of 6,000 gallons of ethanol per acre per year. Algenol selects cyanobacteria strains and enhances their ability to produce ethanol by overexpressing fermentation pathway enzymes, allowing each cell to channel carbon into ethanol production. Algenol uses a proprietary photobioreactor system to cultivate cyanobacteria and collect ethanol. The method involves a marine strain of algae and therefore can use seawater. It also has the added benefits of consuming carbon dioxide from industrial sources and not using farmland.	Algenol’s proprietary flexible plastic film photobioreactor (PBR). Capital costs to construct its patented facility will range between $4.00 and $6.00 per annual gallon of capacity. A pilot-scale integrated biorefinery in Florida on 36 acres was broken ground in 2011.	Algenol intends to produce 1 billion gallons annually by 2012. The company says its production costs will be less than $1.00 per gallon (sale for $3.00 per gallon). Algenol’s goal is 20 billion gallons per year of low — cost ethanol by 2030.
Florida, USA Ethanol
Sapphire 2007 Energy	Headquarters in San Diego, USA; green crude farm in New Mexico, USA	A liquid that has the same composition as crude oil	Sapphire produces "green crude," a liquid that has the same composition as crude oil. The company has shown that its fuel can be used in	The company uses open ponds, raceway. The test site in Las Cruces, NM, at 22 acres, has more than 70 active ponds, varying in size from	The first phase of Sapphire’s Green Crude Farm was operational in August 2012. When completed, the facility will produce 1.5 million

Company Founded Name Time Location Biofuel Type Technology Cultivation Equipments Market
two commercial flights (Continental and JAL airlines) and a cross­country road trip (Algaeus).	14-foot test ponds to 300- foot, 1-million-liter production ponds. The green crude farm located in Columbus, NM, will have 300 cultivated acres.	gallons per year of crude oil and consist of approximately 300 acres of algae cultivation ponds and processing facilities. The plan is to make 1 million gallons of diesel and jet fuel per year by 2011, 100 million gallons by 2018, and 1 billion gallons per year by 2025. Seambiotic’s Algae Plant in China was finished in late 2011 with raceway ponds on approximately 10 hectares. Seambiotic believes that this plant is able to produce enough algae biomass to convert into fuel at prices competitive with traditional fuel by 2012.
Biodiesel and bioethanol	Seambiotic grows microalgal cultures in open ponds using flue gases such as carbon dioxide and nitrogen from a nearby coal plant as feedstocks. The 1,000-square-meter facility produces roughly 23,000 grams of algae per day. Three tons of algal biomass would yield around 100 to 200 gallons of biofuel.
Seambiotic 2003	Israel	Open ponds, raceway.
South San Francisco, USA	Solazyme’s proprietary microalgae are heterotrophic, grow in the dark in fermenters, and are fed plant sugars.	Standard industrial fermentation equipment.	In 2010, Solazyme delivered over 80,000 liters of algal-derived biodiesel and jet fuel to the U. S. Navy. Subsequently, Solazyme was awarded another contract with the U. S. Department of Defense for production of up to 550,000 additional
Biodiesel
Solazyme 2003

Company Founded Name Time Location Biofuel Type Technology Cultivation Equipments Market
liters of naval distillate fuel. Solazyme went public (IPO) in 2011 at $18 per share and raised $198 million in the process. In 2012, Solazyme expected to archive a 2-million-liter annual capacity. Solix’s demonstration facility performed at over 3,000 gallons of algae oil per acre per year in 2010.
Solix 2006 Colorado, Biodiesel BioSystems USA	Solix uses a proprietary closed photobioreactor system and claims that the system can produce up to seven times as much biomass as open-pond systems. The algal oil is extracted through the use of chemical solvents such as benzene or ether. Solix is also collaborating with the Los Alamos National Laboratory to use its acoustic-focusing technology to concentrate algal cells into a dense mixture by blasting them with sound waves. Oil can then be extracted from the mixture by squeezing it out; this makes the extraction process much easier and cheaper, obviating the need for chemical solvents.	Photobioreactor system includes Solix’s proprietary Lumian panels, Solix Lumian Algae Growth System (AGS™). Solix’s demonstration plant has three algae cultivation basins totaling 3/4 of an acre (0.3 hectares). The plant has over 150,000 liters of algae under cultivation.

FIGURE 2.5 The process of Algenol’s Direct to Ethanol® technology (www. algenolbiofuels. com/media/media- gallery).

media preparation, harvesting, reinjection, and system cleaning. Before 2009, the introduction of the Lumian AGS system especially mentioned the vertical orientation of panels that can provide "extended surface area." However, according to the pictures on Solix’s Website, the panels now are horizontally arranged. The AGS panels contain tubes that deliver CO2 as a carbon source and deliver air to remove oxygen (a byproduct of photosynthesis). According to an article of the IOP Conference Series in 2009 (Willson, 2009), the marginal cost of large-scale production using the AGS system was approximately $1/liter ($150/barrel), with a defined path of reducing the production cost by half over the next two to three years.

Sapphire and Seambiotic both choose raceway open ponds to cultivate their algae. Sapphire releases very little technology information about its process: "We grow the algae in open ponds with only sunlight, CO2, and nonpotable saltwater in deserts" (see Figure 2.8a). Seambiotic also grows microalgal cultures in raceway open ponds using flue gases carbon dioxide and nitrogen from a nearby coal plant as the feedstock (see Figure 2.8b). Seambiotic has carried out an R&D pilot study comprising about a 1,000-meter square of ponds in an Israel power plant to use the flue gas to cultivate algae. Both companies emphasize the low cost of using open ponds and choose marine algae strains to reduce biotic contamination.

Solazyme’s algal cultivation method is much different from those of the previously men­tioned companies. Solazyme uses large fermentation tanks to incubate algae in the dark and feed them plant sugars. This platform makes the feedstock more flexible, and it is able to use

2.5

COMMERCIAL MICROALGAE CULTIVATION SYSTEMS FOR BIOFUEL PRODUCTION

FIGURE 2.6 The flexible plastic film photobioreactors used by Algenol; A) the structural diagram, B) the appear­ance (www. algenolbiofuels. com/media/media-gallery).

low-cost sugars, varying from sugarcane to corn stover, woody biomass, switchgrass, and other cellulosic materials. By this heterotrophic incubation, algae can accumulate more oil in cells. According to data shown on Solazyme’s Website, the oil content in the company’s algae cells is in excess of 80% (see Figure 2.9). Considering that the average wild alga yields only 5-10% oil content, this enhanced yield is very critical to lowering the production cost of biofuels.

The Solix Lumian AGS4000 system (www. solixbiofuels. com/content/products/lumian-ags4000).

FIGURE 2.8 (a) Sapphire’s green crude farm with raceway open ponds (www. sapphireenergy. com/rendition. medium/images/multimedia/green%20crude%20farm%20ponds. jpg). (b) Seambiotic’s pilot plant (www. seambiotic. com/uploads/Seambiotic%20Ltd.%20-%20Algae%20Pilot%20Plant%20white%20paper. pdf).

2.6

CONCLUSIONS

FIGURE 2.9 Solazyme’s heterotrophic algae cultivation platform (http://solazyme. com/technology).

2.3 CONCLUSIONS

Production of biofuels and other products from microalgae requires a massive amount of microalgae biomass. Effective cultivation technology for large-scale microalgae biomass pro­duction is of great importance in the commercialization of the microalgae-based industry. The growth of microalgae is greatly influenced by environmental conditions, such as light supply, temperature, CO2 supply, and so on. Therefore, an appropriate operating condition to create optimal conditions should be applied for microalgae cultivation. Moreover, the design and configuration of cultivation systems and photobioreactors also play a pivotal role in the mass production of microalgae biomass.

Toward that end, various open and closed cultivation systems have their own pros and cons. In general, closed systems provide better stability and cultivation efficiency, whereas open systems are much cheaper and easier to scale up. As a result, selection of a suitable cul­tivation system is highly dependent on the characteristics of the target microalgae species as well as the climate and environmental conditions of the cultivation site. In addition, since out­door cultivation of microalgae is inevitable for commercial applications, people need to cope with the challenges and limitations arising from the natural environment, such as the avail­ability of sunlight, the limitation of CO2 and nutrient sources, and variations in ambient tem­peratures. Furthermore, a cost and life-cycle analysis should be performed on the developed process to assess economic feasibility as well as environmental impacts.

In the 1950s, the increase in world population and the prediction of insufficient protein supplement for humans led to the search for alternative and unconventional sources of nutrients. Microalgae emerged as candidates for this purpose. Research has been directed to­ward the development of functional products—food additives such as vitamins, antioxidants, highly digestible proteins, and essential fatty acids. Microalgae can supply several of these nutrients, and they have potential health benefits (Cavani et al., 2009; Petracci et al., 2009).

Microalgae are currently used in the form of tablets, capsules, or liquids. These microor­ganisms can be incorporated into pastas, cookies, food, candy, gum, and beverages (Liang et al., 2004). Due to their varying chemical properties, microalgae can be applied as a nutri­tional supplement or as a source of natural proteins, dyes, antioxidants and polyunsaturated fatty acids (Spolaore et al., 2006; Soletto et al., 2005).

The Laboratory of Biochemical Engineering (LEB) at the Federal University of Rio Grande (FURG) in southern Brazil has developed research projects since 1998 that study the cultiva­tion of Spirulina on a pilot scale on the banks of the Mangueira Lagoon, as additives to meals for children of the region. Products that are easy to prepare, store, and distribute and that are highly nutritious and accepted by the consumer have been developed here.

These products include instant noodles, pudding, powdered mixture for cake, cookies, chocolate milk powder, instant soup, isotonic drinks, powdered gelatin, and cereal bars.

These products will be prepared at the Center for Enrichment of Foods with Spirulina (CEAS) located at the university. In Camaqua (Brazil), the company Olson produces organic Spirulina capsules for importation.

4.1.1 Carbon Dioxide’s Role in Photobioreactors

An important issue in most photobioreactors and the first step in CO2 fixation is the diffusion of CO2 from the gas phase to the aqueous phase. The solubility of CO2 in the culture media de­pends on depth of the pond, the mixing velocity, the productivity of the system, the alkalinity, and the outgassing. It has been reported (Becker, 1994) that only 13-20% of the supplied CO2 was absorbed in raceway ponds when CO2 gas was bubbled into the culture fluid as a carbon source. Binaghi et al. (2003) achieved a maximum value of 38% efficiency of carbon utilization in Spirulina cultivation. Gas-liquid contact time and gas-liquid interfacial area are, therefore, two key factors to enhance the gas-liquid mass transfer. In addition, high oxygen tension is problematic, since it promotes CO2 outgassing and competes with CO2 for the CO2-fixing enzyme (RuBisCO).

The capacity for carbon dioxide storage in a growth medium is important because it deter­mines the amount of CO2 that may be used for medium saturation, leading to high growth rates and in-process economics. Since CO2 reacts with water, producing carbonic acid and its anions, chemical equilibrium will have a significant impact on the amount of carbon dioxide stored. pH is the major determinant of the relative concentrations of the carbonaceous system in water and affects the availability of carbon for algal photosynthesis in intensive cultures (Azov, 1982).

The absorption of CO2 into alkaline waters may be accelerated by one of two major uncatalyzed reaction paths: the hydration of CO2 and subsequent acid-base reaction to form bicarbonate ion, and the direct reaction of CO2 with the hydroxyl ion to form bicarbonate. The rate of the former reaction is faster at pH values below 8, whereas the latter dominates above pH 10. Between pH 8 and 10, both are important.

Microalgae can fixate carbon dioxide from different sources, including CO2 from the atmo­sphere, from industrial exhaust gases (e. g. furnaces flue gases), and in form of soluble carbon­ates. Traditionally, microalgae are cultivated in open or closed reactors and aerated with air or air enriched with CO2. Industrial exhaust gases contain up to 15% of carbon dioxide in their composition, being a rich (and cheap) source of carbon for microalgae growth.

In microalgae cultivation, high concentrations of CO2 are not usually used because it may result in decreasing the pH, since unutilized CO2 will be converted to HCO3 . Shiraiwa et al. (1991) and Aizawa and Miyachi (1986) reported that an increase in CO2 concentration of sev­eral percent resulted in the loss of a carbon concentration mechanism (CCM), and any further increase was always disadvantageous to cell growth. Most processes use air enriched with CO2 (2-5% CO2 final concentration), but some studies using high CO2-resistant strains are being described in scientific literature.

If there is not enough CO2 gas supply, algae will utilize (bi)carbonate to maintain its growth. When algae use CO2 from bicarbonate, an increase of pH is observed (a growth in­dicator), even reaching growth-inhibition pH values. To overcome pH fluctuation, the CO2 gas injection should be controlled in such a way that photosynthesis rates are balanced with enough and continuous availability of dissolved carbon. Interesting studies about isolation and selection of strains with high CO2 absorption capacity, which is an important step no matter the process in development, are available in scientific literature. Maintaining constant CO2-free concentration in the media will keep carbon uptake constant.

The ability to accumulate DIC has been shown to occur in many algae and cyanobacteria (Williams and Colman, 1995). Whereas CO2 can diffuse into algal cells and is the substrate for carbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubiscO), it forms a small proportion of the total available inorganic carbon. The largest proportion of total DIC available to microalgae consists of ionic HCO3 , which has a low capacity for diffusion across cell membranes (Young et al., 2001). A number of eukaryotic microalgae have devel­oped mechanisms that permit the use of HCO3 for photosynthesis (Miller and Canvin, 1985). Access to the larger pool of HCO3 is assumed to involve one or both of two basic processes:

1. In some green algae, the use of HCO^ has been correlated with the presence of external carbonic anhydrase (CA) activity (Aizawa and Miyachi, 1986). In these cases external CA is thought to facilitate the use of HCO^ by maintaining equilibrium between HCO^ and CO2, and thereby maintaining the supply of CO2 to a specific transporter (Aizawa and Miyachi, 1986).

2. Direct HCO^ transport via a transmembrane bicarbonate transporter, which has been demonstrated even in cells that have external CA activity (Williams and Turpin, 1987). The involvement of transmembrane ATPase proteins was also reported in DIC uptake by chlorophytes (Ramazanov et al., 1995).