Hong-Wei Yen1, I-Chen Hu2, Chun-Yen Chen3, and

Jo-Shu Chang4

xDepartment of Chemical and Materials Engineering, Tunghai University,

Taichung, Taiwan

2Far East Bio-Tec Co. Ltd., Taipei, Taiwan, Far East Microalgae Ind Co. Ltd.,

Ping-Tung, Taiwan

3Center for Bioscience and Biotechnology, National Cheng Kung University

Tainan, Taiwan

^Department of Chemical Engineering, National Cheng Kung University,

Tainan, Taiwan

1.3 INTRODUCTION

Recently, microalgae have been recognized as a promising platform for biofuels produc­tion and biorefineries. Microalgae have very high growth rates compared with those of ter­restrial plants, thereby demonstrating high CO2 fixation efficiency and high biomass productivity. In addition, a wide range of applications of microalgae also addresses the high potential of commercialization of microalgae-based products, such as biofuels, nutraceuticals, cosmetics, pharmaceuticals, animal and aquacultural feeds, and so on. One of the key technologies that support the development of the microalgae industry is the cultivation of microalgae on a large scale and at low cost. This microalgae cultivation technology is associated with the design of the type and configuration of open or closed cultivation systems and photobioreactors, as well as the identification of the operating con­ditions leading to the optimal growth performance of the target microalgae. In particular, producing biofuels from microalgae requires a massive amount of microalgae biomass. This demand makes the microalgae cultivation technology even more important. In this chapter, the principles and basic knowledge of microalgae growth and mass production are intro­duced. Commonly used cultivation systems and photobioreactors are described. Their advantages and weaknesses are compared. In addition, some examples of the commercial microalgae cultivation process for biofuels production are given to provide updates on the commercial development of microalgae-based biofuels. The limitations and challenges that large-scale microalgae cultivation may face are addressed and discussed.

Dunaliella is abiflagellate unicellular green alga. Cells are round-shaped and found inbrackish environments; it is a motile species and has a high tolerance for salt, temperature, and light. Motion of cells is important since it facilitates nutrient transport, especially in poor-nutrient waters. Dunaliella species are relatively easy to culture. The cell divides by simple binary fission, and no evidence of cell lysis, encystment, or spore formation is observed (Segovia et al., 2003).

Dunaliella thrives over a wide pH range and expresses a capacity for extremely efficient DIC accumulation, incorporating a capacity to use HCO — in addition to CO2 (Aizawa and Miyachi, 1986; Young et al., 2001). Kishimoto et al. (1994) cultivated a Dunaliella strain for pigment pro­duction with 3% of CO2 and achieved a carbon uptake of 313 mg L-1 day-1. Sydney et al. (2011) cultivated a D. tertiolecta strain and achieved a CO2 fixation rate of 272 mg L-1 day-1.

Dunaliella is an important microalgae for industrial processes since it produces a wide variety of commercial products (mainly pigments) and the rupture of the cells is very easy. p-carotene large-scale production facilities are in operation around the world (Hawaii, United States, Australia, Japan).

The biological fixation of CO2 can be carried out by higher plants or microalgae. The sources of CO2 for microalgal cultivation are atmospheric CO2; CO2 from industrial flue gas; and chemically fixed CO2 in the form of soluble carbonates (Kumar et al., 2010). One ki­logram of algal dry cell weight utilizes around 1.83 kg of CO2. Annually, around 54.9-67.7 tonnes of CO2 can be sequestered from raceway ponds, corresponding to an annual dry weight biomass production rate of 30-37 tonnes per hectare (Brennan and Owende, 2010).

The CO2 can be the limiting nutrient in microalgal cultivation if it is available in low con­centration in the feed gas (when air is used as a source of CO2) or when mixing is not suffi­cient. However, the high CO2 concentration causes a reduction in pH, which can inhibit the growth of some microalgae (Wang et al., 2012).

Open tanks may have a limited carbon source due to the low transfer of mass. The simple bubbling of CO2 in the cultures may not be sufficiently effective, because the residence time of the bubble can be very short and is lost to the atmosphere. In these cases a high concentration of free CO2 must be maintained through direct injection of the flue gas from power plants, cement, and petrochemical factories during cultivation.

The biofixation of CO2 can be increased while maintaining an alkaline pH, because this will accelerate the absorption of gas through two reactions: CO2 hydration and subsequent acid — base reaction to form HCOg and direct reaction of CO2 with OH — to form HCOg (Amaro et al. 2011). The most common system employed for pH control is the on-off type system in which CO2 is injected into culture when the pH exceeds a predefined set point (Kumar et al., 2010).

The engineering of a photobioreactor must also be designed to add gas transfer equipment, which will increase the gas distribution and the contact of the gas with the liquid. Some of these items include mechanical systems (propellers, blades, and brushes), coarse and fine bubble diffusers (perforated piping, slotted tubes, discs, or domes), jet aerators, aspirators, U-tubes, and hollow fiber membrane modules (Kumar et al., 2010).

In vertical tubular, horizontal, or airlift photobioreactors, the biofixation of the CO2 is increased by the route of the gas along the tube as well as by the use of sprinklers that release small bubbles, increasing the contact surface between the gas and the liquid.

The fixation of CO2 by microalgae has received attention due to the production of biomass with potential application in the production of biofuels, reducing the emission of greenhouse gases and participating in the treatment of effluents (Kumar et al., 2010).

A vertical column photobioreactor is made up of vertical tubing (glass or acrylic) that is trans­parent to allow the penetration of light for the autotrophic cultivation of microalgae. A gas sparger system is installed at the bottom of the reactor; it converts the inlet gas into tiny bub­bles, which provide the driving force for mixing, mass transfer of CO2, and removing O2 pro­duced during photosynthesis (Figure 2.2). Normally, no physical agitation system is implemented in the design of a vertical column photobioreactor. Vertical tubular

photobioreactors can be categorized as bubble column or airlift reactors based on their liquid flow patterns inside the photobioreactor.

Bubble column reactors are cylindrical vessels with height greater than twice their diameter. They are characterized by low capital cost, high surface-area-to-volume ratio, lack of moving parts, satisfactory heat and mass transfer, relatively homogenous culture environment, and efficient release of O2 and residual gas mixture (Loubiere et al., 2009). The gas bubbling up­ward from the sparger provides the required mixing and gas transfer. Therefore, the sparger’s design is critical to the performance of a bubble column. In scale-up of the photobioreactor, perforated plates are adopted as the sparger used in tall bubble columns to break up and re­distribute coalesced bubbles (Janssen et al., 2000). Light supply for autotrophic cultivation often comes from outside the column. Nevertheless, an inner-illumination design is gradually becoming acceptable due to higher light-penetration efficiency and more uniform light dis­tribution (Loubiere et al., 2009). Photosynthetic efficiency greatly depends on gas flow rate as well as the light and dark cycle created when the liquid is circulated regularly from central dark zone to external zone at a higher gas flow rate.

Airlift reactors, common in traditional bioreactor designs, are made of a vessel with two interconnecting zones. One of the tubes, called a gas riser, is where the gas mixture flows

Air

compressor

upward to the surface from the sparger. The other region, called the downcomer, does not re­ceive the gas, but the medium flows down toward the bottom and circulates within the riser and the downcomer. Based on the circulation mode, the design of an airlift reactor can be fur­ther classified into one of two forms: internal loop or external loop (Loubiere et al., 2009). The riser is similar to that designed for a bubble column, where the gas moves upward randomly and haphazardly. An airlift reactor has the advantage of creating flow circulation where liquid culture passes continuously through dark and light phases, giving a flashing-light effect to the microalgal cells. Residence time of gas in various zones controls performance, affecting parameters such as gas-liquid mass transfer, heat transfer, mixing, and turbulence. A rectangular airlift photobioreactor is also suggested to have better mixing characteristics and high photosynthetic efficiency, but the design complexity and difficulty in scale-up both are disadvantages.

The drying process is one of the major limitations in the production of low-cost commod­ities (fuel, food, feed) and high-value products (p-carotene, polysaccharides). The process to be selected depends on the final product desired. The use of dehydration increases the shelf life of the biomass as well as the final product.

Several methods have been used to dry Chlorella, Scenedesmus, and Spirulina. Some of the most widely used methods include spray drying, drum drying, freeze drying, and sun drying (Richmond, 2004). Due to the high water content, the sun-drying method is not efficient to transform humid biomass into powder. The spray-drying method is not economically feasible for low-value products such as biofuels and protein (Mata et al., 2010).

Eduardo Bittencourt Sydney, Alessandra Cristine Novak, Julio
Cesar de Carvalho, Carlos Ricardo Soccol

Biotechnology Division, Federal University of Parana, Curitiba, Brazil

4.1 INTRODUCTION

The Framework Convention on Climate Change, signed in Rio de Janeiro in 1992, made global warming a major focus, and the development of technologies for reducing/absorbing greenhouse gases (GhG) gained importance. After another 20 years, at Rio + 20, the final document stated clear concern about emissions and the need to reduce them by 2020.

Rubin et al. (1992) divided the GhG reduction alternatives into three groups: conservation, direct mitigation, and indirect mitigation. Conservation measures reduce electricity con­sumption and thus GhG emissions; direct mitigation techniques capture and remove CO2 emitted by specific sources; and indirect mitigation involves offsetting actions in which GhG producers support reductions in GhG emission.

The concept behind most disposal methods is to offset the immediate effect on the levels of carbon dioxide in the atmosphere by relocation, i. e., by injection into either geologic or oceanic sinks (Stewart and Hessami, 2005). Relocation in ocean and deep saline formations has the capacity for 1012 tons of CO2, whereas global carbon dioxide emissions in 2009 were 33 x 106 tons (Olivier et al., 2011), which means 30,000 years of relocation. Problems related to this issue are the unknown possible environmental problems (such as acidification, for example), costs, and the necessity to concentrate CO2 before relocation (how will it work to transport CO2 emissions, for example?).

Therefore, long-term mitigation technologies for CO2 and other GhG gas removal came to be developed. They can be generally classified into two categories: (1) chemical reaction — based technologies and (2) biological CO2 mitigation.

Chemical reaction-based CO2 mitigation approaches are energy-consuming and costly processes (Lin et al., 2003), and the only economical incentive for CO2 mitigation using the chemical reaction-based approach is the CO2 credits to be generated under the Kyoto Protocol (Wang et al., 2008). For example, CO2 can be instantly absorbed through bubbling it in a hy­droxide solution at 40°Celsius, producing sodium or ammonium bicarbonate. However, the demand for these salts (although high—equivalent to around 14Gt/year) is supplied by the Solvay process, whereas a CO2 process would require previous synthesis of sodium hydroxide.

Biological CO2 mitigation has attracted a good deal attention as a strategic alternative. Microalgae cultivation gained importance because it associates CO2 mitigation and produc­tion of a wide range of commercial bioproducts.

Despite the fact that the existence of microalgae has been known for a long time, studies for its use as industrial microorganisms are relatively recent. Initial studies of microalgae culti­vation began in the late 1940s and early 1950s for its potential as a source of food. Concerns about water pollution in the 1960s increased interest in the use of microalgae in wastewater treatment. The perception in the 1970s that fossil fuels would run out made these microorgan­isms a focus of renewable fuel production. In the 1980s microalgae were used as a source of value-added products, specifically nutriceuticals. In the late 1980s the low cost of oil caused a loss of interest in microalgae-based energy, whereas research with nutraceuticals and biomass for feed continued. In the 2000s, global warming concerns associated with high oil prices made microalgal bioenergy projects popular again.

To create microalgal products, it is necessary to develop mass-cultivation techniques and to understand the physiological characteristics of each strain. There have been extensive stud­ies on process optimization (media and physicochemical parameter optimization, screening and isolation of high CO2 tolerants, search for new valuable products, optimization and development of new vessels and systems for cultivation, for example) to try to overcome the economic issues faced in industrial-scale production of microalgae. Two other aspects are gaining importance: the use of industrial residues (to reduce media costs) and the carbon market (carbon credits as an additional element in the economic evaluation of the process).

The evaluation of nutrient needs in microalgal cultures is an important tool in process development using residues (domestic or industrial), and the quantification of carbon dioxide fixation is of great industrial interest since carbon credits can be traded on the international market and companies may use the process as a marketing strategy.

The rate of carbon uptake is limited by the metabolic activity of microalgae, which is in turn limited by photosynthesis. The ability to identify rates of consumption of nutrients is thus of considerable importance to the understanding of the metabolism of microalgae and to avoid problems in industrial cultivation of such microorganisms.

Jorge Alberto Vieira Costa* and Michele Greque de Morais

*Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering,
Federal University of Rio Grande, Rio Grande, RS, Brazil

1.1 INTRODUCTION

Microalgal biotechnology has emerged due to the great diversity of products that can be developed from biomass. Microalgal biomass has been industrially applied in areas such as dietary supplements, lipids, biomasses, biopolymers, pigments, biofertilizers, and biofuels. To produce these compounds, microalgae can be grown using carbon dioxide and industrial wastes, which reduces the cost of culture medium nutrients and alleviates the environmental problems caused by these effluents. However, the high cost of production of microalgal bio­mass (compared to agricultural and forestry biomasses) is one of the major barriers that must be overcome in order for their industrial production to be viable.

Although efforts have been directed at the optimization of the medium and processes, the development of cultivation systems that are cost-effective and highly efficient must be signif­icantly improved for large-scale production to be viable (Wang et al., 2012; Wang and Lan, 2011). Microalgal cultivation on a large scale has been studied for decades (Lee, 2001).

The first unialgal cultivation was carried out with the microalga Chlorella vulgaris by Beijerinck in 1890, who wanted to study the physiology of the plants (Borowitzka, 1999). Dur­ing World War II, Germany, using open ponds, increased algal cultivation for use as a food supplement. With the onset of industrialization, some study groups at the Carnegie Institute in Washington, D. C., implemented algae cultures for carbon dioxide biofixation. In 1970 Eastern Europe, Israel, and Japan began commercial production of algae in open ponds to produce healthy foods (Ugwu et al., 2008).

Open pond cultivation systems are the most industrially applied because of their low cost of investment and operational capital. This system’s major difficulties are the control of operating conditions, which can cause low biomass productivity, and the control of contam­inants, which can be excluded by using highly selective species (Shu and Lee, 2003).

Compared to open ponds, closed photobioreactors may have increased photosynthetic ef­ficiency and higher production of biomass (Wang et al., 2012). However, closed photobioreactors have a high initial cost, and only microalgal strains with specific physiol­ogies may be used (Harun et al., 2010), which is why different types of closed photobioreactors have been developed in recent decades (Wang et al., 2012).

The objective of this study was to present the advantages and disadvantages of open ponds compared to other photobioreactors as well as to examine factors that affect the cultures and the bioproducts obtained.

1.3.1 Carbon Sources

Carbon sources are usually the most critical factors for the growth of microalgae. In gen­eral, microalgae can be grown under photoautotrophic, heterotrophic, and mixotrophic con­ditions using diversified carbon sources, such as carbon dioxide, methanol, acetate, glucose, or other organic compounds (Xu et al., 2006). Photoautotrophic cultivation means that microalgae use inorganic carbon (e. g., carbon dioxide or bicarbonates) as the carbon source to form chemical energy through photosynthesis (Ren et al., 2010). Some microalgae species can directly use organic carbon as the carbon source in the presence or absence of a light sup­ply. This is called heterotrophic cultivation (Chojnacka and Noworyta, 2004). However, the most commonly used carbon source for microalgae growth and biofuels production is still carbon dioxide or bicarbonates, since using organic carbon sources would be too expensive for producing low-price products such as biofuels.

In addition, from the aspect of CO2 emissions reduction, a net-zero CO2 emission could be achieved when the biofuels are directly converted from using CO2 as the substrate. In partic­ular, photoautotrophic growth of microalgae represents an ideal model of reutilization of CO2 coming from flue gas of power plants and industrial activities (Packer, 2009), as microalgae biomass can be further utilized to produce biofuels or other value-added products (Hsueh et al., 2007; Raoof et al., 2006). In addition, most microalgae have much higher cell growth and CO2 fixation rates than terrestrial plants (around 10-50 times higher), which demon­strates another advantage of direct conversion of photoautotrophic growth of microalgae.

Therefore, it seems more reasonable from the perspectives of economic feasibility and environmental protection that microalgae-based biofuels should be produced via photoautotro­phic growth of microalgae. However, another thought is to produce biofuels from microalgae grown under heterotrophic conditions using organic carbon sources (e. g., sugars) derived from biomass. In this way, biofuel productivity could be markedly enhanced, since heterotrophic growth of microalgae is usually faster than autotrophic growth (Chen, 1996). Nevertheless, again, the high cost of obtaining the organic carbon sources from raw biomass is still a great concern.

Temperature is one of the major factors that regulate cell morphology and physiology as well as the byproducts of the microalgal biomass. A high temperature generally accelerates the metabolism of microalgae and a low temperature can inhibit growth (Munoz and Guieysse, 2006).

The optimum temperature for growth varies among species of microalgae (Ono and Cuello, 2003). High temperatures during the day have a favorable effect on growth rates due to photosynthesis. High temperatures at night are not desired in microalgal cultivation due to the increased respiration rate; they result in a high expenditure of cellular energy and consequent reduction of cellular concentration.

The temperature also influences other factors that are important for cultivation, such as the ionic balance of water, pH, and solubility of O2 and CO2. Different species of microalgae are affected by temperature at different levels (Park et al., 2011). In the case of combustion gases emitted in power plants, the gas temperatures reach 120 °C. In this case, the rate of CO2 biofixation may depend on the installation of a heat exchange system or the use of thermo­philic species. The solubility of O2 and CO2 increases the temperature and results in the fix­ation of high concentrations of O2 by oxigenase of RuBisCO. Thus the affinity for RuBisCo by CO2 decreases with increasing temperature (Kumar et al., 2011).

The temperature of cultivation in the photobioreactor is determined by the air temperature, the duration of solar radiation, and the relative humidity of air. The depth and the surface of the culture and the material of construction of the photobioractor are factors that stabilize the temperature of the culture. Mechanisms of temperature control cause significant changes in the design of a photobioreactor. With no temperature control, a closed photobioreactor can reach values of 10-30 °C above ambient temperature. Some mechanisms of temperature control in closed photobioreactors include immersion of the culture in water, spraying with water, shading, or incorporating a heat exchanger with the photobioreactor (Wang et al.,

2012) . In raceway-type photobioreactors, the temperature is generally greenhouse controlled. At low temperatures the greenhouse is kept closed, maintaining the temperature. On hot days the sides of the greenhouse can be erected, thus reducing the temperature in the inner area where the raceways are located.

Flat panel photobioreactors feature important advantages for mass production of photoauto­trophic microorganisms. The simple flat plate photobioreactor consists of vertically translu­cent flat plates, which are illuminated on both sides and stirred by aeration (Figure 2.3). This simple building methodology for glass flat plate reactors provides the opportunity to easily construct reactors with any desired light path. Light is evenly emitted from a flat transparent surface screen or from lamps above the culture. The plate surface is usually made of glass or optical light film, and the circulation is achieved by the same means of rising air bubbles, as

with the tubular systems. However, flat plate systems may also experience problems with relatively high space requirements, high light energy requirements, difficulties in cleaning, and possible low efficiency in terms of mass production per unit of space (Slegers et al.,

2011) . The productivity of flat-plate photobioreactors is highly dependent on the space requirements between the panels and the areal productivity constraint for outdoor applica­tion. On the other hand, if the flat plate systems are to be operated indoors, then some crucial factors would be involved, including distance of light sources from panels, temperature effects, illumination of one or both panel sides, light path, and so on. Scale-up of the flat plate system is potentially difficult due to the increase of hydrostatic pressure with the increase of volume. In general, the structure of flat plate systems cannot tolerate very high pressure. Moreover, the hydrodynamic stress on microalgae cells may affect the microalgae growth. In addition, the biomass productivity in parallel flat panels is strongly influenced by shading and light penetration between the panels (Posada et al., 2012). To further reduce the equip­ment cost, a novel design of a vertical flat panel photobioreactor, consisting of a transparent bag (i. e., plastic) located on a rigid frame, has been proposed and could greatly enhance the economic feasibility (Tredici and Rodolf, 2004).