Photosynthates provide an endogenous source of acetyl-CoA by activated acetyl-CoA synthetase in the stroma, from free acetate, or from the cytosolic conversion of glucose to pyruvate during glycolysis (Somerville et al., 2000; Schwender and Ohlrogge, 2002). This acetyl-CoA is preferentially transported from the cytosol to the plastid, where it is converted to the fatty acid and subsequently to TAG, which again is transported to the cy­tosol and forms the lipid bodies (Figure 8.1). The acetyl-CoA pool will be maintained through the Calvin cycle, glycolysis and pyruvate kinase (PK) mediated synthesis of py­ruvate from PEP, which occur in the chloroplast in addition to the cytosol. The first reaction of the fatty acid biosynthetic pathway towards the formation of malonyl-CoA from acetyl — CoA and CO2 is catalyzed by the enzyme Acetyl-CoA carboxylase (ACCase). (Ohlrogge and Browse, 1995). Figure 8.2 illustrates the conversion of acetyl-CoA to malonyl-CoA by utilizing ATP. During this process, seven molecules of acetyl-CoA and seven molecules of CO2 form seven molecules of malonyl-CoA. This malonyl Co-A undergoes synthesis of long carbon-chain fatty acids through repeating multistep sequences, as represented in Figures 8.2 and 8.3. A saturated acyl group produced by this set of reactions becomes the substrate for subsequent condensation with an activated malonyl group (Ohlrogge and Browse, 1995).

FIGURE 8.3 Sequential chain elongation steps and formation of precursor molecules (palmitic acid) from CO2

Kuan-Yeow Show1, Duu-Jong Lee2

xDepartment of Environmental Science and Engineering, Fudan University,

Shanghai, China

^Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan

9.1 INTRODUCTION

Extensive effort is being made globally to exploring renewable energy sources that could replace fossil fuels in mitigating global warming and other environmental issues. Hydrogen is a promising fuel alternative to conventional fossil fuels because it releases energy ex­plosively without air pollutants in combustion. Most of the hydrogen in use currently is produced through thermochemical processes via electricity generation from fossil fuels. Because the current hydrogen fuel is based on the use of nonrenewable fossil-fuel resources, a major issue related to conventional hydrogen production is sustainability.

Biohydrogen production is deemed a key development in creating a sustainable energy supply and a promising alternative to fossil fuels. Hydrogen production via biological pro­cesses is carried out largely at ambient temperatures and pressures and hence is less energy intensive than chemical or electrochemical ones. As a desired green energy product of natural bioconversion, biohydrogen metabolism is primarily the domain of bacteria and microalgae. Within these groups, it involves many taxonomically diverse species, a variety of enzymes, and metabolic pathways and processes (Schulz, 1996; Vignais et al., 2001; Weaver et al., 1980). Biological processes use the enzyme hydrogenase or nitrogenase as a hydrogen-producing protein. This enzyme regulates the hydrogen metabolism of prokaryotes and some eukaryotic organisms, including green algae. The function of nitrogenase as well as hydrogenase is linked with the utilization of metabolic products of photosynthetic reactions that generate reductants from water.

Current development of algal hydrogen production is focusing on biophotolysis and photosynthesis-hydrogen production using various microbial species. Sunlight is necessary for hydrogen production by photosynthetic microorganisms. Photoautotrophic green microalgae and cyanobacteria use carbon dioxide and sunlight as the respective sole carbon and energy sources. The reducing power for cellular photosynthesis and/or biophotolysis comes from water oxidation under light irradiation (Ghirardi et al., 2000; Schutz et al.,

2004) . This chapter examines the perspectives and state-of-the-art of algal hydrogen research in the context of pathways of hydrogen production, bioreactor design and operation, and eco­nomic evaluation. Prospects and challenges in algal hydrogen production are also outlined.

Besides iodine, compounds derived from halogens are produced by red and brown macroalgae (Butler and Carter-Franklin, 2004). Halogenated compounds appear as several classes of primary and secondary metabolites, including indoles, terpenes, acetogenins, phenols, fatty acids, polyhalogenated monoterpenes, and volatile halogenated hydrocarbons (e. g., bromoform, chloroform, and dibromomethane) (Dembitsky and Rozentsvet, 1990; Butler and Carter-Franklin, 2004). In many cases, they possess biological activities of pharmacological interest, as emphasized in Table 10.6. These compounds may also play

multifunctional ecological roles (Suzuki, Takahashi et al., 2002; Brito, Cueto et al., 2002). These kinds of compounds can be extractable by SFE or/and using solvents (Pourmortazavi and Hajimirsadeghi, 2007) or by pressurized liquid with solid-phase extraction (Onofrejova, Vasickova et al., 2010).

To overcome the limitations of the open pond system in algae cultivation, closed photobioreactors are designed to ensure that algal cells are always grown under optimal con­ditions with high consistency in biomass productivity. Since the conditions in a closed photobioreactor system are strictly controlled, the contamination level in the cultivation me­dium is minimized. This permits the cultivating of single algal strain for a prolonged period, and water sources may be reutilized for subsequent cultivation cycles (Brennan and Owende, 2010; Chisti, 2007). Closed photobioreactors are a more flexible system than the raceway pond because the photobioreactors can be optimized according to the biological and physiological characteristics of the algal strain that is being cultivated (Mata et al., 2010). For example, cultivation pH, temperature, CO2 concentration, mixing intensity, and nutrient level can be manipulated to suit the optimal growing conditions of different algal strains.

These advantages have attracted the interest of many researchers to further improve on the operating conditions of closed photobioreactors for commercial-scale implementations. Depending on the algal strains and cultivation conditions, a closed photobioreactor always offers high biomass productivity, generally in the range of 0.05-3.8 g/L/day (Brennan and Owende, 2010). Several types of closed photobioreactor designs, such as flat plate, tubular, and column, are discussed in Table 12.2. For comparison purposes, the characteristics of a raceway pond are also included in Table 12.2.

Recently, a few LCA studies have been performed to evaluate the overall energy balance for cultivating algal biomass in raceway ponds and airlift tubular closed photobioreactors, as shown in Table 12.3. From the table, we see that the airlift tubular photobioreactor can achieve high biomass productivity compared to the raceway pond, but the energy input to operate the entire system was approximately 350% higher than for the raceway pond. Despite the advan­tages of low contamination and minimum water loss due to evaporation, the airlift tubular photobioreactor consumed a huge amount of electricity to power heavy-duty pumps so that

TABLE 12.2 Various Photobioreactor Designs for Algal Cultivation. (Chisti, 2007; Mata et al., 2010; Sierra et al., 2008; Ugwu et al., 2008; Xu et al., 2009)

sufficient mixing and optimum gas-liquid transfer rate are attained. Cultivating algae using the airlift tubular photobioreactor could easily lead to a negative energy balance in producing algal biofuels if no precautionary steps are taken to reduce the energy input. Furthermore, the energy input does not include the energy used for artificial lights during the nighttime, harvesting and drying of algal biomass, water treatment, lipid extraction, and biodiesel conversion. If these factors are taken into consideration, the overall energy balance for culti­vating algae for biofuel production is expected to be even more negative, as revealed by Stephenson et al. (2010) and Razon and Tan (2011) (Table 12.1). Other photobioreactor de­signs, such as column type and flat plate, are relatively low cost compared to airlift tubular photobioreactors, making them more feasible for commercialization. However, more exten­sive research is required to improve the CO2 transfer and mixing in these photobioreactors with minimum energy input.

As already pointed out, growth rates, biomass composition, C/N ratios, fertilizer require­ments, and energy content of the algae are correlated parameters and hence should not be set according to independent assumptions. We advocate for the definition of chemical properties of each biochemical compartment of the algae (e. g., carbohydrates, lipids, membrane) in or­der to justify the fertilizer budget, the energy content of the raw algae, and the extraction res­idue. This would hopefully reduce the spread of values for very important parameters such as nutrient requirement, lipid content, or growth rate.

The most important parameter considered for the development and utilization of a specific type of reactor for microalgae cultivation is the light diffusion. The productivity of photo­autotrophic cultures is primarily limited by the supply of light and suffers from low energy-conversion efficiencies caused by inhomogeneous distribution of light inside the cultures (Grobbelaar, 2000). At culture surfaces, light intensities are high, but absorption and scattering result in decreasing light intensities and complex photosynthetic productivity profiles inside the cultures (Ogbonna and Tanaka, 2000). High light intensities at culture
surfaces may cause photoinhibition, and the efficiency of light energy conversion into biomass (photosynthetic efficiency) is low. An overdose of excitation energy can lead to pro­duction of toxic species (e. g., singlet oxygen) and to photosynthesis damage (Janssen, 2002)

By minimizing depth, volume is reduced or area is increased, light diffusion is maximized, and so is cell concentration. From common types of photobioreactors, light paths in open ponds are usually 10-30 cm depth, in tubular reactors ranges from 1-5 cm, and in flat panel reactors from 2-5 cm.

The light regimen itself is influenced by incident light intensity, reactor design and dimension, cell density, pigmentation of the cells, mixing pattern, and more. In outdoor photobioreactors the light regimen is also influenced by geographical location, time of day, and weather conditions. Nowadays, open paddlewheel-mixed pond is the most commonly used photobioreactor.

Some studies discuss the effect of mixing and productivity due to the "flashing-light" effect: A few milliseconds’ flashes of high light intensity followed by a several-fold longer period of darkness do not reduce culture productivity from those under constant illumination (Kok, 1953). This effect is not observed in ponds, where the light/dark period is longer. For example, although light/dark cycles of 94/94 ms were sufficiently short to increase the pho­tosynthesis efficiency in cultures of Dunaliella tertiolecta, light/dark cycles of 3/3 s were too long and the PE decreased in comparison to continuously illuminated cultures (Janssen et al.,

2001) . This refers to the theory of photosynthesis, in which carbon fixation is not dependent on the presence of light because sufficient energy has been absorbed.

Microalgae are more efficient than higher plants with respect to photosynthesis, through which light, together with CO2, is converted to chemical energy. Aside from photo­autotrophy, some microalgae are capable of growing heterotrophically as well as mixotro — phically. Heterotrophy refers to the fact that microalgae utilize organic carbon as the solo carbon and energy source for their reproduction in the absence of light; mixotrophy is indic­ative of microalgae performing growth in the presence of light through use of both CO2

(photosynthesis) and organic carbon sources. A number of microalgae have been reported for heterotrophic growth, among which green algae, in particular, Chlorella, are the most studied (Table 6.1). Microalgae are capable of utilizing a wide range of organic carbon sources, includ­ing sugars, hydrolyzed carbohydrates, waste molasses, acetate, and glycerol, as well as or­ganic carbons from wastewater (Table 6.1). Regardless of the microalgal species and strains, sugar—in particular, glucose—is the most commonly used organic carbon for boosting heterotrophic growth of microalgae (Table 6.1).

The uptake of external glucose relies on a hexose/H+ symport system that has been char­acterized in Chlorella (Hallmann and Sumper, 1996). In the presence of glucose, the hexose/H+ symport system is activated and transports glucose and H+ (1:1) into cytosol at the cost of equal ATP molecules (Tanner, 2000). The catabolism of transported glucose starts with a phos­phorylation of the hexose to form glucose-6-phosphate, an important intermediate product for respiration, storage, and biomass synthesis. Two pathways that share the initially formed glucose-6-phosphate are proposed to be involved in the aerobic glycolysis in algae—namely, the Embden-Meyerhof-Parnas (EMP) pathway and the pentose phosphate (PP) pathway (Figure 6.1; Neilson and Lewin, 1974). Both pathways are present in cytosol and contribute to the glucose metabolism in algae of autotrophy, mixotrophy, and heterotrophy, though their contributions may vary largely (Yang et al., 2000, 2002; Hong and Lee, 2007). For instance, glucose is mainly metabolized via a PP pathway in heterotrophic Chlorella pyrenoidosa, which accounts for 90% of total glucose metabolic flux distribution (Yang et al., 2000). The dominant role of a PP pathway was also demonstrated in the heterotrophic culture of the cyanobacterium Synechocystis sp. PCC6803 (Yang et al., 2002). In contrast, the EMP pathway serves as the major flux of glucose metabolism in algae in the presence of light (Yang et al., 2000,2002), suggesting the regulation of light on glycolysis. Table 6.2 shows the central metabolic network of glucose in heterotrophic algae with stoichiometric reactions.

As we mentioned, closed cultivation systems that house the growth of algae under con­trolled conditions are referred as photobioreactors (PBRs). Photobioreactors provide a more controlled environment than open ponds because these systems are closed and everything that the algae need to grow (carbon dioxide, water, and light) can be supplied with in the system (Weissman, 1987; Pulz, 2001). There are different types of PBRs reported for algae cultivation. PBRs facilitate better control of culture environment, such as carbon dioxide sup­ply, water supply, optimal temperature, efficient exposure to light, culture density, pH levels, gas supply rate, mixing regime, and so on (Mata et al., 2010). High mass transfer is one of the important criteria for PBR design, especially for CO2 sequestration (Ugwu et al., 2008). Ag­itations in PBR are done either mechanically or nonmechanically. Non-mechanical agitation can be observed airlift, bubble column, tubular reactor, and flat panel operations. PBRs spe­cifically designed for CO2 sequestration have the flexibility of using CO2-rich gas as a means of mixing as well as providing nutrients for the growth of algae (Hu et al., 1996). PBRs can be operated in both batch and continuous modes. In comparison with open culture systems, a closed photobioreactor is easy to control with regard to environmental parameters and can achieve high growth rates (Pulz, 2001; Sierra et al., 2008). Higher biomass of microalgae pro­ductivity is obtained in closed cultivation systems where contamination can also be prevented (Ramanathan et al., 2011). Fully closed photobioreactors provide opportunities for monoseptic culture of a greater variety of algae than open cultivation systems (Borowitzka, 1999). Various types of closed cultivation systems are studied to a great extent.

Chlamydomonas reinhardtii has been comprehensively investigated in terms of potential hydrogen photoproduction; in addition to hydrogen, a variable amount of byproducts are generated as part of the microalgal biomass. Its volatile nature permits the biomass to remain essentially intact (Kruse, Rupprecht et al., 2005).

To obtain further energy, the biomass can itself be processed via extraction and transester­ification of the remaining lipids to produce biodiesel. For example, the total lipid content of the biomass of the C. reinhardtii strain D1 after photobiological hydrogen production is 15 ± 2% on a dry-weight basis; oil may then be extracted, being composed of 3.3% w/woii phytols, 21% w/woii triglycerides, 39% w/woii polar lipids, and 37% w/woii highly polar lipids, to eventually biodiesel composed of 41% saturated fatty esters, 53% mono unsaturated fatty esters, and 7.2% polyunsaturated fatty esters (mainly linoleic acid) (Torri, Samori et al., 2011). This mix of methyl esters adheres to European Union (EU) standard EN 14214 pertaining to biodiesel specifications.

Following biodiesel production from the spent biomass, the lipid-free residue can still be used for animal feed or be anaerobically digested into biogas (Sialve, Bernet et al., 2009) as discussed in further detail in the following paragraph. Moreover, pyrolysis of the residue left after extraction may represent another pathway to produce extra energy (Mohan et al., 2006). Pyrolysis of the microalgal extraction residue may lead to oil with a quality lying between pe­troleum tar and bio-oil from lignocellulosic biomass (Miao et al., 2004); the mass yields of biochar, oil, and gas are 44 ± 1%, 28 ± 2%, and 28 ± 1%, respectively. The ash content of said biochar, obtained via combustion at 700 °C, was 45 ± 5%. On an ash-free basis, the mass yields of biochar, oil, and gas were 24 ± 5%, 38 ± 9%, and 36 ± 1%, respectively (Torri, Samori et al., 2011). Since a major portion of ashes, phosphorus, and nitrogen are retained in biochar, it may be used as fertilizer to improve the productivity of soil, thus contributing to abatement of greenhouse gases while making it possible to convert carbon-neutral energy into carbon­negative bio-energy (Kruse and Hankamer, 2010).

Additionally, production of biogas via fermentation of the microalgal biomass offers the possibility to recycle a large proportion of the original nutrients. Although this option is not economically feasible at low throughput rates, it will become a more interesting possi­bility as medium costs become a larger fraction of the final cost, coupled with consideration of phosphorous limitations (Cordell, Drangert et al., 2009).

In their natural environment, macroalgae grow on rocky substrates and form stable, mul­tilayered, perennial vegetation, capturing almost all available photons. Due to the fact that seaweeds are fixed to their substrate, values for maximum productivity may be 10 times higher for a seaweed stand than for a plankton population and can be as high as 1.8 kg C m~2 y_1. Commercial farming of seaweed has a long history, especially in Asia. The kelp Laminaria japonica is the most important, with 4.2 million tons (Mio. t) cultivated mainly in China (Luning and Pang, 2003). Approximately 200 species of seaweeds are used worldwide, about 10 of which are intensively cultivated, including the brown algae Laminaria japonica and Undaria pinnatifida; the red algae Porphyra, Eucheuma, Kappaphycus, and Gracilaria; and the green algae Monostroma and Enteromorpha (Luning and Pang, 2003).