Dark or heterotrophic fermentation by anaerobes as well as some microalgae, such as green algae on carbohydrate-rich substrates, can produce hydrogen in an anaerobic environment without the need of light energy (Zhang et al., 2006; 2007a, b, c; 2008a, b, c, d; Show et al., 2007; 2010; Lee et al., 2011). The possibility with dark fermentative hydrogen production from algal biomass remains that hydrogen was produced by heterotrophic bacterial satellites present in the algal biomass slurries (Carver et al., 2011; Lakaniemi et al., 2011). It has been well established that methane is generated in conventional anaerobic fermentation in two distinct stages: acidification and methane production. Each stage is carried out by specific microorganisms through syntrophic interactions. Hydrogen is produced in the first-stage

acidogenesis as an intermediate metabolite, which in turn is used as an electron donor by methanogens at the second-stage methanogenesis.

Formation of molecular hydrogen in dark fermentation is generally accom­plished through two pathways in the presence of specific coenzymes (Show et al.,

2011) . One pathway is by a formic acid decomposition route; the other pathway is the reoxidization of nicotinamide adenine dinucleotide (NADH) route represented by NADH + H+ + 2Fd2+! 2H+ + NAD+ + 2Fd+ and 2Fd2+ + 2H+! 2Fd+ + H2 under the mediation of hydrogenase. The Embden-Meyerhof, or glycolytic, pathway is un­doubtedly the most common route for glucose degradation to pyruvate, which functions in the presence or absence of oxygen (Prescott et al., 2002). In this pathway, glucose is converted into pyruvate associated with the conversion of NADH from NAD+ via an­aerobic glycolysis represented by C6H12O6 + 2NAD+! 2CH3COCOOH + 2NADH + 2H+. Electron transfer via pyruvate-ferredoxin oxidoreductase or NADH-ferredoxin oxidore- ductase and hydrogenase could be affected by the corresponding NADH and acetyl-CoA levels or prevailing environmental conditions. Thus, the oxidation-reduction state has to be balanced through the NADH utilization to form several reduced compounds, i. e., lactate, ethanol, and butanol, resulting in a lowered hydrogen yield.

Theoretically, it is possible to harvest hydrogen at the acidogenesis stage of anaerobic fer­mentation if only acidogens are left to produce hydrogen gas and other metabolites and the final methanogenesis stage and other hydrogen-consuming biochemical reactions are inhibited during the dark fermentation. However, inhibition of hydrogen-consuming micro­organisms in complex microbial consortia decomposing algal biomass for hydrogen produc­tion poses a challenging task. It has been reported that the hydrogen produced from green algae C. vulgaris and D. tertiolecta biomass by anaerobic enriched cultures containing BESA was subsequently consumed by nonmethanogenic microorganisms (Lakaniemi et al.,

2011) . Similar hydrogen utilization was also reported from the work on hydrogen production by anaerobic sludge fed with lipid-extracted Scenedesmus algal biomass (Yang et al., 2010).

During the dark fermentation, carbohydrates are converted into hydrogen gas and volatile fatty acids and alcohols, which are organic pollutants and energy carriers. For the purpose of energy production and protection of the water bodies, a second-stage process is necessary to recover the energy residues remaining in the effluent in the form of fatty acids and alcohols. Thus the fermentative reactor becomes part of a process wherein the effluent post-treatment process and hydrogen utilization should also be included. A possible second-stage process is photofermentation, anaerobic digestion, or microbial fuel cells, which have been assessed in a recent review (Show et al., 2012).

For hydrogen produced from dark fermentation to be used alone in an internal combustion engine or a fuel cell, some issues such as biohydrogen purification, storage, and transport are to be addressed. Unlike a biophotolysis process that produces only hydrogen, the gaseous product of dark fermentation is a mixture of primary hydrogen (generally less than 70%) and CO2 but may also contain other gases such as CH4, H2S, ammonia, and/or moisture. Pu­rification of the hydrogen is essential before the hydrogen utilization can be practical (Show et al., 2011; 2012). Nevertheless, hydrogen production by dark fermentation is an attractive process in the sense that it does not demand large land space and is not affected by weather conditions (solar radiation is not a requirement). Also, among the biohydrogen production processes, dark fermentation is deemed to be more favorable. Hydrogen is yielded at a high rate, and various organic compounds and wastewaters are enriched with carbohydrates as the substrate results in low-cost hydrogen production (Hallenbeck and Ghosh, 2009). Hence, the feasibility of the technology yields a growing commercial value.

Rawel Singh, Thallada Bhaskar, Bhavya Balagurumurthy

Biofuels Division, CSIR-Indian Institute of Petroleum, Dehradun, India

11.1 INTRODUCTION

Concerns over energy supply security, global climate change as well as local air pollution, and the increasing price of energy services are having a growing impact on policy making throughout the world. Today’s energy and transport system, which is based mainly on fossil energy carriers, can in no way be evaluated as sustainable. The search for a sustainable and environment-friendly source of hydrocarbons is the need of the hour. Research efforts di­rected toward the conversion of biomass into a liquid transportation fuel have their origins in the first U. S. energy crisis of October 1973, a consequence of the Yom Kippur War and the Organization of Petroleum Exporting Countries (OPEC) oil embargo. Subsequently, the 1979 Iranian revolution and more recent concerns about the security of imported petroleum and the contribution of carbon dioxide (CO2) emissions to global warming trends have led to renewed efforts to provide an essentially CO2-neutral supply of transportation fuel (Blanch, 2012).

It has been long expected that biofuels and biorefineries can at least partially mitigate these problems and create more sustainable and balanced economies. To date three generations of biofuels have been developed. The first-generation biofuels were made from edible feedstock such as corn, soybean, sugarcane, and rapeseed. Biofuel production from these resources was, rightfully or not, blamed for the subsequent surge in food prices. Second-generation biofuels produced from waste lignocellulosic biomass and dedicated lignocellulosic feedstock such as miscanthus, switchgrass, or poplar have advantages over those of the first generation. The main advantages are higher yields and lower land requirement (in both quality and quantity).

The concept of using algae to make fuels was already being discussed 50 years ago (Oswald and Golueke, 1960), but a concerted effort began with the oil crisis of the 1970s. Large research
programs in Japan and the United States focused on developing microalgal energy produc­tion systems. Third-generation biofuel feedstock, micro-, and macroalgae can have an edge over the previous two generations. These marine organisms show the prospect of high bio­mass yields without requiring any arable land and have the potential to be cultivated in con­tainment off-shore (Trent, 2012). Moreover, some algal species grow well in saline, brackish, and waste water, which makes them more promising feedstock than terrestrial crops that rely exclusively on fresh water. These features, along with successful methods for large-scale algae cultivation and processing, can make third-generation feedstock superior to that of pre­vious generations (Daroch et al., 2012).

The typical differences between lignocellulosic biomass and algal biomass are depicted in Figure 11.1. The transition from first — and second-generation to third-generation biofuels offers a reduction in land requirements. This is due to higher energy yields per hectare as we move along this transition as well as utilization of nonagricultural land (Fenton and O hUallachain, 2012). In addition, algae do not deplete any soil nutrients that could aid ag­riculture. Green and blue-green (cyanobacteria) microalgae have been on Earth for millions of years and differ substantially from higher plants. They are single-celled microorganisms that live in aquatic environments, and all components necessary for life and procreation are lo­cated within a single cell. In higher terrestrial plants, specialized cells with specific functions are required, making up roots, stems, flowers, and other functional parts. Cellulose, hemicel — lulose, and lignin often provide structural support for these specialized cells and are present in significant quantities. In contrast, microalgae and cyanobacteria are not lignocellulosic in

hemicellulose, lignin

composition but are composed of proteins, lipids, noncellulosic carbohydrates, and nucleic acids (Heilmann et al., 2010).

Today, efforts are being made to maximize the productivity of biomass and identify new species of plants and processes to fulfill future demands for food, fodder, materials, and en­ergy. The utilization of algae is seen as one of the possible alternatives (Kroger and Muller — Langer, 2012). Algae are a feedstock that has certain advantages over land-based feedstock. Under favorable conditions, the growth rate of algae is estimated to be 5-10 times higher than land-based crops, implying a higher production rate of theoretically convertible biomass. Additionally, certain species may have a high fraction of lipids or carbohydrates of up to 70-80 wt% (Chisti, 2007). There are several reasons for the high production rate. One of them is the higher photosynthetic efficiency.

Many commercial efforts are underway to maximize economic return and improve energy balances in algal cultivation. Currently, much work is focused on extracting high-value chemicals (e. g., nutraceuticals) and energy-dense lipids (e. g., for biodiesel) from algae, but this still leaves behind a large residual of "defatted" biomass. Effective utilization of defatted algal biomass will be necessary to achieve favorable energy balances and production costs (Pan et al., 2010).

The use of macroalgae for energy production has received less attention for the production of fuels/chemicals, despite the fact that macroalgae have long been cultivated for several pur­poses (food production, chemical extraction) in China, Korea, the Philippines, and Japan. The productivity is in the range of 1-15 kg m-2 y-1 dry weight (10-150 tdw ha-1 y-1) for a seven — to eight-month culture. Either brown algae (Laminaria, Sargassum) or red algae have been used so far for such purposes (Aresta et al., 2003).

Apart from biodiesel, bioethanol is another attractive biofuel that is used as a substitute for gasoline. Biomass that contains sugar, starch, or cellulose is used by yeast as a substrate dur­ing the fermentation process, releasing bioethanol as the product and CO2 as the byproduct (Brennan and Owende, 2010). Since algae are able to accumulate significant amounts of carbohydrates (mainly referred to as starch) inside their cells, the potential to utilize the car­bohydrate for bioethanol production is high (Harun et al., 2010). Among the algal strains that have been identified as having high carbohydrate content are Chlamydomonas reinhardtii (53%), C. reinhardtii (45%), Chlorella vulgaris (12-37%), Chlorella sp. (21-27%), and Scenedesmus sp. (13-20%) (John et al., 2011).

Unlike terrestrial plants, algal cells are buoyant and thus do not require lignin and hemi — celluloses for structural support (John et al., 2011). Therefore, carbohydrate extraction from algal biomass is expected to be simpler than carbohydrate extraction from lignocellulosic materials; avoiding the complicated pretreatment steps to remove lignin and the economic feasibility of bioethanol production can consequently be improved. Nevertheless, the extracted algal carbohydrates need to be hydrolyzed further (hydrolysis process) to simple reducing sugars (e. g., glucose) for yeast to effectively convert the sugar into bioethanol during the fermentation process.

Recently, several effective carbohydrate hydrolysis methods for algal biomass, such as using dilute acid solution (Harun and Danquah, 2011), dilute alkaline solution

(Harun et al., 2011), and enzymatic methods (Choi et al., 2010; Harun and Danquah, 2011), have been reported in the literature, as shown in Table 12.5. The data in the table show that the bioethanol yields are comparable to the yields attained from sugar and lignocellulosic feedstocks, indicating that it is technically viable to produce bioethanol from algal biomass. Reutilization of the algal residue after lipid extraction for bioethanol conversion instead of using the freshly dried algal biomass is also possible (Lam and Lee, 2011). This would be a more realistic approach because two different types of biofuels are simultaneously produced from the algal biomass, and thus the life-cycle energy balance of algal biofuels can be further strengthened.

This concept has been proven viable in a recent study in which lipids from Chlorococum sp. were preextracted with supercritical CO2 at 60 °C and subsequently subjected to fermentation with the yeast Saccharomyces bayanus (Harun et al., 2010). From the study, algal biomass with pre-extracted lipids gave 60% higher bioethanol concentration for all samples than the dried algal biomass without lipid extraction. This is because during supercritical CO2 extraction of lipids, the algal cell wall is ruptured due to the high temperature and pressure required for the supercritical process (Harun et al., 2010). The rupturing of the cell wall leads to the release of carbohydrates and subsequently being hydrolyzed to simple reducing sugar. Thus the algal residues after lipid extraction are readily available for fermentation with yeast. Based on this study, a maximum bioethanol yield of 3.83 g/L was achieved from 10 g/L of lipid-extracted algal residue.

To assess the microalgae biomass production cost of any process, it is necessary to know the complete process flowchart in detail, including a list of equipment and equipment size in addition to raw material uptake and energy consumption (Kalk and Langlykke, 1986).

Figure 14.2 summarizes the steps necessary to define the major contributions to production cost: (1) depreciation, (2) raw materials and utilities, and (3) labor and supervision. The total production cost is calculated as the sum of depreciation plus direct production costs (raw ma­terials and utilities, along with labor and supervision). From the block diagram of the process (the conceptual approach), a detailed process flowchart can be defined based on production capacity and kinetic parameters of the different unit operations performed. The flowchart al­lows us to know the type and size of equipment necessary as well as the mass and energy

FIGURE 14.2

balances on the entire process. The cost of major equipment can be obtained from the sup­pliers or, alternatively, from bibliographic references or databases. From this information, the total fixed capital is calculated, multiplying by the corresponding Lang factors according to the nature of the item. The value of these factors is available for a wide variety of processes, values for microalgae-based processes being previously verified (Acien et al., 2012a). The de­preciation includes not only amortization of the fixed capital, which is a function of the esti­mated lifetime, but also the property tax, insurance, and purchase tax.

The direct production cost includes raw materials, utilities, labor, and others (supervision, maintenance, tax, contingencies, etc.). The amount of raw materials required is calculated from mass balances according to the specified flowchart, whereas the consumption of utilities is calculated from the power and water use of the process. The cost of raw materials has to include transport to the facility and the market values obtained from suppliers. With regard to power, the cost of electricity can vary according to consumption and energy required; there­fore, a detailed analysis of different suppliers is recommended.

Water is an important utility for microalgae production; thus its cost needs to be accurately determined. Water cost is a function of its quality (seawater, brackish water, freshwater, wastewater) and uptake volume. Moreover, in this section the cost of wastewater treatment of effluents from the facility has to be included. Whatever the quality of water used, the cost of pumping the water into and out of the facility has to be included in the power consumption item, separate from the power required to operate the facility, which is mainly related to wa­ter recirculation in the photobioreactors.

Labor consists of the workers necessary to correctly operate the process and the general costs of supervision and management, in addition to maintenance, taxes, and contingencies. To determine the direct production cost, it is necessary to know the cost of the raw materials, power, water, and labor, whereas the other costs are calculated by previously defined factors. The labor cost varies widely as a function of personnel qualification levels and facility location. Supervision and other costs are calculated based on the number of personnel directly involved in the operation of the facility and their salaries. Therefore, by reducing the number or salary levels of direct personnel, the labor and supervision cost greatly reduces.

Following this methodology, it is possible to ascertain the production cost of microalgae biomass for any facility. Moreover, the production cost at any other scale can also be approx­imated simply by modifying the cost of major equipment according to the scale chosen and
then multiplying the direct cost by the adequate factor in order to increase the production capacity. The process or equipment cost can be scaled up or down from a basic size using an exponential law for which a value of 0.85 is considered appropriate. This equation is not valid for large-scale changes because a certain technology can be feasible at one scale but might not be available on a larger scale. Thus, a maximum scale-up factor of 10 is consid­ered acceptable without revising the technology. Whenever larger requirements are needed, the scale-up has to be solved by multiplying the number of units.

SizeB 085

CostB = CostA S_l_ (14.1)

SizeA

SpiruIina is a filamentous cyanobacterium recognized mainly by its multicellular cylindri­cal arrangement of trichomes in an open helix along the entire length (Vonshak, 1997). Under the microscope, it appears as blue-green filaments of unbranched cylindrical cells, in helical trichomes. The filaments are movable and move freely around its axis, and they are not heterocystic. They are up to 1 mm in length; the cell diameter ranges from 1-3 gm in small species and 3-12 gm in the larger species (Richmond, 1990).

This microalga inhabits various media such as soil, sand, swamps, alkaline lakes and brackish, marine, and fresh water. Through photosynthesis, it converts nutrients into cellular matter and releases oxygen. The components needed for cell growth are water, a carbon source, nitrogen, phosphorus, potassium, magnesium, iron, and other micronutrients.

In natural lakes, the limited supply of nutrients may regulate the growth cycles, and the cell density increases rapidly, reaches a maximum concentration, and retreats when nutrients are depleted. The release of nutrients from dead cells or the supply of nutrients initiates a new cycle (Henrikson, 1994).

There are many controversies in the morphology and taxonomy of cyanobacteria of the genera Spirulina and Arthospira. Many studies have described the properties of Spirulina max­ima and Spirulina platensis, and both species are considered to be of the genus Arthospira and not Spirulina. The differences between the genera have been based on the G + C content of DNA and lipid profile (Romano et al., 2000).

The helical shape is only maintained in liquid medium; in solid medium the filaments take a spiral shape, and the transition from the helical shape to the spiral shape is slow, whereas the opposite takes place instantaneously. Most species of Spirulina present a granular cyto­plasm containing gas vacuoles and septa that are easily visible. Electron microscopy reveals that the cell wall of Spirulina platensis is probably composed of four layers.

The life cycle of Spirulina begins when a trichome (filament consisting of cells) elongates, and this is followed by an increase in the number of cells as a result of repeated interspersed cell divisions. The microalga cell fragmented into several parts by the formation of special­ized, lysis-promoting necridic cells, which give rise to small chains (two or four cells) called hormogonia, which develop into new trichomes. The number of cells in the hormogonium increases by cellular fission, while the cytoplasm becomes granulated and the cells take on a bright bluish-green color. Due to this process, trichomes increase in length and take their typical helical shape (Richmond, 1990).

In both lab-scale and pilot-scale microalgae cultivation systems, the key factors that need to be considered for the design and operation of microalgae cultivation systems are as follows: (1) how to use appropriate light sources (intensity and wavelength), (2) how to enhance light conversion efficiency, and (3) how to maintain an appropriate microalgae biomass concentra­tion during prolonged operation. In addition, the stability of continuous culture of microalgae is usually poor, because the cell growth and target-product production are sensitive to changes in the environment and the medium composition.

Maintaining a sufficient cell concentration in the continuous microalgae cultivation system is also a challenge. Therefore, many large-scale outdoor microalgae cultivation systems are operated in a semibatch mode, in which a portion of microalgae culture is harvested within a specific cultivation time period and an equal amount of fresh medium is refilled into the cultivation system. In addition, most commercial-scale microalgae cultivation is carried out in open ponds, since solar light energy is directly utilized. Therefore, there are challenges such as contamination by other microorganisms or alien microalgae species, direct exposure to ultraviolet (UV) irradiation, low light intensity or uneven light energy distribution (Kim et al., 1997), day-night cycles, diurnal variation, and requirements for large areas of land (Laws et al., 1986). Moreover, since the intensity of sunlight varies greatly with the seasons, solar spectrum, and operating time, it is very difficult to maintain steady microalgae culture performance in outdoor cultivation.

The limitation of light energy is also one of the most commonly encountered problems in large-scale cultivation when the size of the microalgae cultivation system is increased. In this case, the illumination area per unit volume is often considered as a design criterion. The fac­tors mentioned here greatly limit the light conversion efficiency and productivities of outdoor microalgae cultivation systems. Other factors that may also lower the biomass productivity are consumption of biomass by respiration in the dark zones of the reactor, insufficient mixing of CO2 and nutrients, and the mechanical damage due to the shear stress on the algal cells. Variation in biomass concentration and composition (e. g., carbohydrate or lipid content) may occur when different culture media and operation modes are used.

Despite the fact that good production performances of target products can be achieved using lab-scale microalgae cultivation systems, there are still very few successful commercial-scale processes. This is mainly because of the higher operating costs, unstable light intensity, and lower mixing efficiency when the microalgae are grown outdoors on a large scale. Consequently, appropriate operating configurations with innovative design of microalgae cultivation system are required to achieve commercially viable production of microalgae biomass and target products.

Therefore, highly efficient light sources and good circulation devices are the key to pro­mote microalgae cell growth in the design of commercially feasible microalgae cultivation systems. If the light source has a narrow spectral output that overlaps the photosynthetic ab­sorption spectrum of microalgae, the emission of light at unusable wavelengths would be eliminated, thereby improving the overall energy conversion. Among the available light sources, light-emitting diode (LED) is the only one that meets the foregoing criteria. LEDs are an economic external light source that is energy-saving and small enough to fit into any microalgae cultivation system. They also have a very long life expectancy, and their elec­trical efficiency is so high that heat generation is minimized. LEDs have a half-power band­width of 20-30 nm, which can match photosynthetic needs. On the other hand, circulation is also very important in the outdoor microalgae cultivation system. The benefits include keep­ing microalgae in suspension, decreasing heat generation within the microalgae cultivation system, uniform distribution of the cells and the liquid broth, improving CO2 mass-transfer efficiency, and degassing the O2 produced during photosynthesis. Therefore, the develop­ment of economically successful production of microalgae biomass requires the improvement of both light efficiency and mixing efficiency for microalgae growth at low cost.

The Kyoto Protocol invented the concept of carbon emissions trading in a flexible mech­anism whereby developed countries could use carbon credits to meet their emission reduc­tion commitments. The world carbon market is based on a cap-and-trade system. According to Mark Lazarowicz (2009), under cap-and-trade, a cap is set on emissions, as explained fur­ther by the author: "Allowances are provided, either through purchase or allocation, to emit­ters covered by the cap. These emitters are required to submit allowances equal to the amount of greenhouse gases emitted over a predetermined period. The difference between expected emissions and the cap creates a price for the allowances. Emitters who can reduce emissions for less than the price of an allowance will do so. If, however, abatement costs more than the price of an allowance, it makes sense to purchase the allowance. The transfer of allowances is the ‘trade.’ The relative difficulty of abatement or scarcity of allowances sets the price of carbon. In theory, those that can reduce emissions most cheaply will do so, achieving the reduction at the lowest possible cost." For this reason, the carbon market seems to be a tem­porary alternative while cleaner technologies are developed, including new ones and improvement of the existing ones.

The carbon market jumped from $63 billion in 2007 to $126 billion in 2008, which means almost 12 times the value of 2005, according to the World Bank report of 2009. Credits were sold for 4.8 billion tons of carbon dioxide, a value 61% higher than that of the previous year. By 2020 the market could be worth up to $2-3 trillion per year (Point Carbon, Carbon Market Transactions in 2020: Dominated by Financials?, May 2008).

The world carbon market is mainly dependent on energy-use policies. The focus is to replace existing high dependence on fossil fuels with renewable ones; around 90% of total global CO2 emissions are from fossil fuel combustion (excluding forest fires and woodfuel use; Olivier et al., 2011). The principal technical means of reducing fossil fuel consumption (and conse­quently emissions) are substituting fossil fuels with renewable or less carbon-content sources of energy and improving energy efficiency. Renewable energy’s share of the global energy supply increased from 7% in 2004 to over 8% by 2009 and 2010 (Olivier et al., 2011).

According to the "Long-term trend in global CO2 emission, 2011 report," total global CO2 emissions had increased 30% since 2000, to 33 billion tones, and 45% since 1990, the base year of the Kyoto Protocol. In 1990 the industrialized countries, with a mitigation target for total greenhouse gas emissions under the Kyoto Protocol (including the United States, which did not ratify the protocol), had a share in global CO2 emissions of 68% versus 29% for developing countries. In 2010 the large regional variation in emission growth trends resulted in shares for 54% of developing countries and 43% for mature industrialized countries.

Microalgae can play a very interesting role in this context. While fixating carbon during growth (to be traded in the market), some species can accumulate lipids, which can be use for direct combustion or transformed in biodiesel to replace fossil sources. This is one of the developing technologies that receives more attention from the scientific community around the world.

The carbon market for microalgal carbon mitigation processes is a big challenge. Its en­trance in this market will coexist with other renewable energy technologies that are receiving lots of investment, which means that it must be more advantageous or differentiated. Trades of carbon papers are carried mainly based on agriculture and forestry (reforestation, land management, reduced emissions from deforestation). Great efforts are being made in the de­velopment and implantation of renewable energy technologies (wind power, solar photovol­taic, and vegetable-based biodiesel technologies).

In terms of development of more efficient and sustainable industrial processes, microalgae can play an interesting role through combining the use of domestic and industrial wastewater (mainly that lacking fermentable carbon) and industrial gaseous wastes with cogeneration of valuable products, reducing carbon emissions and generating tradable carbon papers. According to the mass balance (Equation 4.3), where the biomass composition is given as CH178 No.15Oo.52 (analysis made in CHNS analyzer carried at the Bioprocess Engineering and Biotechnology Department, Federal University of Parana, Brazil), around 1.8 gCO2 is con­sumed for each gram of dry biomass produced during microalgal growth. This means that, for producing 1 Carbon Paper (1 ton CO2), an area less than 1,000 square meters is needed (considering a biomass concentration in the culture of 3 g L-1 and a pond with 20 cm high of liquid).

0. 815 H2O + CO2 + 0.15 HNO3 ! CH1.78 N0.15O0.52 + 1.37 O2 (4.3)

Continuous cultivation with cell recycling, denoted as perfusion culture, is a culture technique combining the advantages of both fed-batch and continuous culture systems, namely, avoiding the substrate inhibition and the inhibition caused by toxic metabolites produced by accumulated algal cells while maintaining high cell density and productivity

(Chen and Johns, 1995; Wen and Chen, 2002a). As illustrated by Figure 6.5b, in a perfusion culture system the algal cells are retained by a retention device, whereas the spent medium (cell-free) was removed from the bioreactor; at the same time, fresh medium was fed into the bioreactor to maintain sufficient nutrient supply. Wen and Chen (2002a) used the perfusion culture system to investigate the heterotrophic production of N. laevis. By employing an ex­ponential feeding of glucose and manipulating the rates of glucose feeding and spent me­dium perfusion, the optimal glucose concentration in the feed was determined to be 50 g L-1 (Figures 6.7a and 6.7b). With the feeding of optimized glucose concentration (S0 = 50 g L-1), a high cell density of 40 g L-1 was achieved in the perfusion culture of N. laevis (Figure 6.7c). Together with the relatively simple setup and operation as well as high biomass

yield coefficient based on glucose, the perfusion culture system potentially may be used to grow algae for heterotrophic production of bio-oils.

A modified perfusion culture system that introduces cell bleeding during perfusion oper­ation was also developed for heterotrophic production of algae (Figure 6.5c; Wen and Chen, 2001b). This system could potentially improve the biomass productivity but at the same time lower the cell density, e. g., from 40 g L-1 to less than 20 g L-1 (Wen and Chen, 2001b; Wen and Chen, 2002a).

It is worth mentioning that different algal species/strains may favor different culture systems to achieve maximized cell density, biomass productivity, and oil productivity. An experimental optimization is required for a selected algal strain to demonstrate which culture system is best for the heterotrophic production of oils. Regardless of the algal strain selected and culture system used, the key to optimizing a production system rests with the cost balance of output and input from a cost-effectiveness point of view.

A coiled transparent and flexible tube of small diameter with separate or attached degassing unit is the basis for the helical type of bioreactor. A centrifugal pump is used to drive the culture through a long tube to the degassing unit. CO2 gas mixture and feed can be circulated from either direction, but injection from the bottom gives better photosynthetic efficiency (Morita et al., 2001). A degasser facilitates removal of photosynthetically produced oxygen and residual gas of the injected gas stream. This system facilitates better CO2 transfer from gas phase to liquid phase due to a large CO2 absorbing pathway (Watanabe et al., 1995). The energy required by the centrifugal pump in recirculating the culture and associated shear stress limits this reactor’s commercial use (Briassoulis et al., 2010). Fouling on the inside of the reactor is another disadvantage of this system.

Alginates are polymers extracted from the cell walls of various brown algae, particularly the species Laminaria, Saccharina, Macrocystis, and Ascophyllum. They are composed of D-mannuronic acid and L-guluronic acid monomers, available in both acid and salt forms; the latter constitutes 40-47% of the dry weight of this brown algal biomass (Arasaki and Arasaki, 1983; Rasmussen and Morrissey, 2007).

Alginates are commonly applied as intermediate feedstock in the food and pharmaceutical industries as stabilizers for the preparation of emulsions and suspensions in ice cream, jam, cream, custard, lotions, and toothpaste but also as coatings for pills. Furthermore, they have found application in the production of paint, construction materials, glue, and paper as well as in the oil, photo, and textile industries (Radmer, 1996).

Besides these technological functions, alginates possess bioactivities, as depicted in Table 10.2. Positive favorable dietary effects of alginates upon faecal microbial fauna have been claimed, as well as prebiotic features (Wang, Han et al., 2006); for instance, the bioactive food additive Detoxal containins calcium alginate and exhibits antitoxic effects on hepatitis. Additionally, mannuronate surfactants derived from alginate have been applied in cosmetics, health products, and agrochemicals (Benvegnu and Sassi, 2010).