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).

To optimize the photosynthesis rate and gas solubility in the media, mixing is very impor­tant. Besides that, mixing is important for homogeneous distribution of cells, metabolites, and heat and to transfer gases across gas-liquid interfaces. Mixing can be done mechanically by paddlewheel in raceways (Figure 4.4) or by gas flow in bubble columns.

FIGURE 4.4 Paddlewheel mixing of raceway ponds at Ouro Fino Agronegcicio (Brazil).

In the heterotrophic batch cultures, high initial concentration of substrates, e. g., sugars, is usually used to provide sufficient carbons for obtaining high cell density. Accompanying the high substrate concentration, however, is the occurrence of possible growth inhibition. For instance, the optimal sugar concentration for growing C. zofingiensis was reported below 20 g L-1, above which the inhibition of algal growth was observed (Ip and Chen, 2005; Liu et al., 2012a). The substrate-based inhibition caused not only the decreased specific growth rate but also the lowered biomass yield coefficient based on sugars (Sun et al., 2008; Liu et al., 2012a), contributing accordingly to the increased cost input. To overcome the inhibition issue associated with batch cultures, fed-batch cultivation is a commonly used strategy in which the substrate is fed into the algal cultures step by step to maintain it sufficiently for cell growth but below the level of inhibition threshold. There have been a number of reports employing fed-batch strategy to grow algae heterotrophically with the aim of avoiding the possible inhibition caused by the initial high substrate and improving the production poten­tial of biomass as well as of oils (Xu et al., 2006; Li et al., 2007a; Sun et al., 2008; Xiong et al., 2008; Liu et al., 2010; 2012a; De la Hoz Siegler et al., 2011; Yan et al., 2011; Chen and Walker,

2012) . Liu et al. (2010) investigated the heterotrophic oil production by C. zofingiensis using fed-batch cultures in a 3.7-L bioreactor. A two-stage feeding was adopted: three times of feed­ing with glucose-containing nutrients (to maintain linear growth) followed by four times of glucose feeding alone (to further increase biomass and induce oil accumulation; Figure 6.4). Glucose concentration of the cultures was maintained between 5 and 20 g L-1. The maximum lipid yield and lipid productivity achieved in the fed-batch cultures were 20.7 g L-1 and 1.38 g L-1 day-1, respectively, representing around a 2.9-fold increase of the those obtained in batch cultures.

Although the employment of fed-batch culture strategy proves able to eliminate the sub­strate inhibition, it cannot overcome the inhibition caused by the toxic metabolites that would be produced by the algal cultures and accumulate as the cells build up, preventing further enhancement of cell density.

FIGURE 6.4 (A) Growth and glucose consump­

tion and (B) lipid production in a two-stage fed-batch A» fermentation of C. zofingiensis in a 3.7-L fermentor. (O) cell biomass; (□) glucose concentration; (column) lipid content; (△) lipid yield; (#) glucose-containing medium feeding; (##) glucose feeding alone. Adapted E from Liu et al. (2010) and the permission for reprint requested.

6.5.1 Continuous Cultivation

The term continuous cultivation refers to the fresh medium being continuously added to a well-mixed culture while cells or products are simultaneously removed to keep the culture volume constant. It allows the steady state of kinetic parameters such as specific growth rate, cell density, and productivity and is thus considered an important system for studying the basic physiological behavior of heterotrophic algal cells. Figure 6.5a shows the schematic di­agram of the continuous cultivation system. This system is capable of effectively eliminating the metabolite-driven inhibition. There are several reports of continuous cultivation of algae in both photoautotrophic (Molina Grima et al., 1994; Otero et al., 1997) and heterotrophic (Wen and Chen, 2002b; Ethier et al., 2011) growth modes. Ethier et al (2011) investigated the continuous production of oils by the microalga Schizochytrium limacinum with various di­lution rates (D) and feed glycerol concentrations (S0). The yields and productivities of bio­mass, total fatty acids (TFA), and docosahexaenoic acid (DHA), shown in Figure 6.6, were over the range of D from 0.2 to 0.6 day-1 (S0 fixed at 90 g L-1) and the range of S0 from 15 to 120 g L-1 (D fixed at 0.3 day-1). The highest biomass productivity is 3.9 gL-1 day-1, obtained with the 0.3 day-1 of D and 60 g L-1 of S0 (Figure 6.6b). The maximum productivities of both TFA and DHA were also achieved at the same D but with a higher S0 of 90 g L-1 (Figures 6.6d and 6.6f). Liu et al (2012a) surveyed the feasibility of using a semicontinuous C. zofingiensis culture fed with waste molasses for oil production. The waste molasses contains relatively high levels of metal ions and salt that are inhibitory to algal growth, causing the

FIGURE 6.5 Schematic diagram of (A) continuous, (B) perfusion, and (C) perfusion­bleeding culture systems. X, cell concentration; V, culture volume; S, carbon concentration in medium; F, flow rate of feed; F1, flow rate of per­fusion; F2, flow rate of bleeding; S0, carbon con­centration in feed. The flow rates are controlled to keep the culture volume constant.

failure of molasses-based fed-batch cultivation when molasses was not pretreated; in con­trast, C. zofingiensis in the semicontinuous culture fed with diluted raw molasses showed comparable growth rate and sugar utilization to that with pretreated molasses (Liu et al., 2012a). Although continuous cultivation can promote the productivity, it is worth to mention that accompanying the increase of dilution rate is the drop of cell density as well as of sub­strate utilization efficiency (Wen and Chen, 2002b). From a cost-effectiveness point of view, this is undesirable in that the residual substrate is wasted with the effluent and more energy input is required to harvest the diluted cells.

FIGURE 6.6 Algal growth, TFA and DHA production of continuous Schizochytrium limacinum in a 7.5-L fermentor with various dilution rates (D) (A, C, E; S0 = 90 gL-1) and feed glycerol concentrations (S0) (B, D, F; D = 0.3 day-1). Adapted from Ethier et al. (2011) and the permission for reprint requested.

It has been reported that with flat panel/plate photobioreactors, high photosynthetic effi­ciencies can be achieved (Hu et al., 1996; Richmond, 2000). Accumulation of dissolved oxygen concentrations in flat plate photobioreactors is relatively low compared to horizontal tubular photobioreactors. Milner’s (1953) work paved the way to the use of flat culture vessels for cultivation of algae. Flat panel photo bioreactors were used extensively for mass cultivation of different algae (Tredici and Materassi, 1992; Hu et al., 1996; Zhang et al., 2002; Hoekema.,

2002) . Lack of temperature control and gas engagement zones are some of the inherent dis­advantages observed with this type of photobioreactor.

The group of phycocolloid polymers, commonly termed hydrocolloids because they are soluble in water, includes alginates, carrageenans, and agars—and red and brown macroalgae have long been used for the production of such compounds (Carlsson, 2007). These polymers are either located in the cell walls or within the cells where they serve as storage materials (Tseng, 2001).

Hydrocolloids account for the major industrial products derived from algae (Radmer, 1996; Pulz and Gross, 2004). They possess several useful properties for the food industry in thickening agents, forming gels and water-soluble films that are commonly applied to sta­bilize such products as ice cream, toothpaste, and mayonnaise (Tseng, 2001), thus taking ad­vantage of their forming a gel upon cooling (Carlsson, 2007). Each major subgroup is described in further detail in the follow subsections.

Conventional hydrothermal treatment processes are divided into three categories: batch — type reactor, semibatch reactor, and continuous reactor.

In a batch reactor, water and reactant are sealed in the same reactor. The reactor is heated from outside or inside. Due to the easy handling and operation of a batch reactor, many re­sults and analysis data in various operation conditions have been reported. But productivity in a batch system does not meet commercial demand. Steel batch autoclaves are used in most cases. Steel autoclaves have the disadvantage of heating slowly, and thus some time is re­quired to reach reaction temperature (Manarungson et al., 1990). Other reactor types include capillaries and tubular steel reactors. Quartz capillaries have also been used as batch microreactors.

In a semibatch reactor, a reactor is filled with reactant and hot compressed water is introduced to the reactant separately. Temperature control of the slurry and flow rate control of the hot water are simple, and moreover product is obtained continuously. However, reactants have to be refilled in the reactor for continuous production. Sakaki et al. developed a semibatch system (Sakaki et al., 1998), but productivity was still very low.

There are two methods in a continuous system; one is a separate type, and the other is a slurry type. Feeding of solid feedstock into a high-pressure reactor is the biggest challenge to the operation of the separate process. On the other hand, a commercial high-pressure slurry pump is available for continuous feeding of high-concentration slurry (Kobayashi et al., 2011). For continuous operation, tubular steel reactors are often used. Other types of reactors, such as the stirred tank reactor, can be used in principle, but to date this configuration has not yet been applied (Navarro et al., 2009).

The nutrient requirement is known to depend on the species but also on the stress that has been induced to stimulate lipid or carbohydrate storage. The nitrogen and phosphorus quota can strongly vary during a starvation period (Geider and La Roche, 2002). The hypotheses on required fertilizers strongly vary according to the species and between the publications for the same species (Lardon et al., 2009; Stephenson et al., 2010; Yang et al., 2011). Needs in ni­trogen vary from 10.9 g kgDM-1 (Lardon et al., 2009) to 20.32 g kgDM-1 (Stephenson et al.,

2010) in limiting conditions and from 9.41 g kgDM-1 (Kadam, 2002) to 77.6 g kgDM-1 (Clarens et al., 2011) without stress. Needs in phosphorus vary from 2.4 g kgDM-1 (Lardon et al., 2009) to 2.58 g kgDM-1 (Khoo et al., 2011) in limiting conditions, and from 0.02 g kgDM-1 (Kadam, 2002) to 71 g kgDM-1 (Yang et al., 2011) without stress. All the au­thors agree on high nutrient consumption for the culture of microalgae, but they differ on the ways to provide them (see Table 13.3). Some authors, such as Sander and Murthy (2010) and Clarens et al. (2010,2011), consider that the needs in nitrogen and phosphorus can be totally or partially covered by the addition of wastewater to the growth medium. But in most of the publications, nutrients are provided by chemical fertilizers. To reduce the nutrient consump­tion, several authors suggest recycling the digestates resulting from the anaerobic digestion of oilcakes (Stephenson et al., 2010; Brentner et al., 2011; Campbell et al., 2011; Clarens et al.,

2011) or of bulk microalgae (Clarens et al., 2011; Collet et al., 2011).

Figure 13.4 shows environmental impacts of various fertilizer sources. As previously dem­onstrated for the energy mix, the source of nutrients can have important consequences on the environmental balance of the energy production from microalgae. Climate-change impact and endpoint impacts on human health and ecosystem can vary by a factor of two, based on the chosen nitrogen fertilizer. For these three impacts, ammonium nitrate is the worst one, and the impacts of ammonium sulphate, calcium nitrate, and urea are quite the same. Concerning resource consumption, urea is the worst, mainly because of the high amount of natural gas used for its production. Clarens et al. (2010, 2011) and Sander and Murthy (2010) suggest using wastewater to grow algae. This assumption allows reducing the con­sumed quantities of freshwater and chemical fertilizers. However, mineral elements’ content in wastewater can strongly vary depending on the place and the period of the year. For these reasons, from our point of view it seems very difficult to rely on such fertilizers.

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.