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.

Phenols (sometimes called phenolics) are a class of chemical compound consisting of a hy­droxyl group (-OH) directly bound to an aromatic hydrocarbon group. The simplest of this class is phenol, the parent compound used as disinfectant and for chemical synthesis. Phlorotannins are an extremely heterogeneous group of phenolic compounds in terms of structure and degree of polymerization; accordingly, they provide a wide range of biological activities (Holdt and Kraan, 2011). Green and red macroalgae possess low concentrations of phenols (Mabeau and Fleurence, 1993) compared to brown macroalgae that are particularly rich in phlorotannin. Typical phenolic contents vary from 1-14% of dry macroalga biomass. Such polyphenols as fucol, fucophlorethol, fucodiphloroethol G, and ergosterol as well as phlorotannin are abundant in brown macroalgae and possess strong antioxidant effects. The concentration of polyphenols exhibits seasonal variations and shows a significant time correlation with the algal reproductive state, besides being affected by a number of other parameters such as location and salinity (Holdt and Kraan, 2011).

Polyphenols entail a cosmetic and pharmacological value owing to their antioxidative activity; they also have shown other favorable effects, e. g., protection from radiation as well as antibiotic and antidiabetic qualities. Several of these effects were tested in bacteria, cell cultures, rodents, and even humans, namely with regard to sexual performance and desire. Certain polyphenols may work as preventative medicines due their several bioactivities (see Table 10.6); in particular, phlorotannins are candidates for development of unique natural antioxidants for further industrial applications in functional food, cosmetic, and pharmaceu­tical formulations (Li, Qian et al., 2009).

For their extraction, several methods can be applied using Soxhlet-based solvent extraction or ultrasonic extraction, as discussed elsewhere (Mahugo Santana, Sosa Ferrera et al., 2009).

Harvesting of algal biomass refers to the separation of algae from water for subsequent biofuel production. The process consists of two distinctive steps: (1) bulk harvesting, to separate algae from bulk suspension via gravity sedimentation, flocculation, and flotation, and (2) thickening, to concentrate the algal slurry after bulk harvesting using techniques such as centrifugation and filtration (Brennan and Owende, 2010; Chen et al., 2011). Harvesting of algal biomass is extremely challenging because of algae’s small cell size (gen­erally 1-20 pm) and suspension in water (Lam and Lee, 2012; Suali and Sarbatly, 2012). The mass ratio of algal biomass to water is considered very low, even if the algae are cultivated in a closed photobioreactor (Chen et al., 2011). For example, the mass ratio of algal biomass to wa­ter lies in the range of 0.00035-0.027 for algae cultivated in a closed photobioreactor, assuming a biomass productivity of 0.05-3.8 g/L/day and cultivated for seven days. When the algal cultivation system (typically a closed photobioreactor) is scaled up for mass production of algal biomass, an average of 73 tonnes of water need to be processed when harvesting 1 tonne of algal biomass. This amount of water is quite substantial; thus, developing effective algal harvesting methods is exceptionally important to strengthen the possibility of commercia­lizing algal biofuel production. Table 12.4 summarizes the current available algal biomass harvesting technologies.

A recent LCA study revealed that current technologies for harvesting and drying algal biomass consumed a significant amount of energy input to produce algal biodiesel (Sander and Murthy, 2010). The study assessed two types of algal thickening methods (without prior bulk harvesting), namely, filter press and centrifugation, and reported that each method contributed 88.6% and 92.7%, respectively, to the total energy input for the LCA. Thus,

harvesting algal biomass using solely centrifugation or filtration is still far from commercial application because of the high energy consumption and high operating cost.

On the other hand, bulk harvesting methods such as flocculation offer an alternative approach to harvesting algal biomass with lower energy input and at a reasonable cost. Conventional flocculants, such as ferric chloride (FeCl3), aluminium sulphate (Al2(SO4)3), and ferric sulphate (Fe2(SO4)3) (Brennan and Owende, 2010), which are widely used in waste­water treatment plants, can be used to agglomerate algal cells to become dense flocs (slurry) and subsequently settle out of the cultivation medium (de Godos et al., 2011). After the flocculation process, water that is still retained in the algal slurry can be concentrated further through centrifugation or filtration (Suali and Sarbatly, 2012).

Nevertheless, conventional flocculants that are always referred to as multivalent salts could contaminate the algal biomass and may affect the quality of the final product. Although no scientific work or assessment has been carried out to justify this claim, flocculant toxicity should not be ignored, especially if health-related products are to be extracted from algal biomass before the algal biomass is diverted to biofuel production. Other organic polymeric flocculants that are biodegradable and less toxic offer an alternative and environmentally friendly way to harvest algal biomass, but these organic polymeric flocculants require further development prior to application on the commercial scale.

After concentrating the algal slurry to 5-15% dry solid content through centrifugation or filtration, further dehydration or drying of the slurry is necessary to facilitate subsequent biofuel production (Brennan and Owende, 2010; Lam and Lee, 2012). The presence of water could severely inhibit the biofuel processing and conversion, including lipid extraction using

chemical solvents and biodiesel production through transesterification (Ehimen et al., 2010). The water would cause some difficulty in recovery of chemical solvents as well as biodiesel refining, requiring even higher energy input for subsequent water separation.

Several dehydration methods are currently applicable to drying the algal slurry, including solar drying, spray drying, freeze drying, and fluidized bed drying (Brennan and Owende, 2010; Desmorieux and Decaen, 2005; Orset et al., 1999; Prakash et al., 1997). Solar drying is apparently the most inexpensive dehydration method because it is free, but a large drying surface is required, and it is time-consuming (Prakash et al., 1997). Nevertheless, solar drying is not feasible in temperate countries where sunlight is not always available throughout the year (Lam and Lee, 2012). Thus, the use of heat generated from fossil fuels cannot be avoided to ensure that the algal slurry is continuously dried for each cultivation cycle. Some LCA stud­ies have emphasized that a large amount of energy is consumed in drying the algal slurry, making commercial algal biofuel production even more challenging (Cooney et al., 2011; Lardon et al., 2009; Lohrey and Kochergin, 2012; Sander and Murthy, 2010; Xu et al., 2011). For example, Sander and Murthy (2010) revealed that using natural gas as the fuel to dry the algal slurry consumed nearly 69% of the overall energy input and consequently resulted in a negative energy balance for producing algal biofuels. Heavy dependence on fossil fuels to dry the algal slurry could reduce the market potential and feasibility for producing algal biofuels; thus, new development of an efficient drying method is required to ensure that the energy input in this step is minimized (Lohrey and Kochergin, 2012).

Experimental studies exploring new technologies to extract energy from algal biomass are often based on lyophilized algae or use solvents that are difficult to use at the industrial scale (e. g., chloroform). For instance, oil extraction performance and oil esterification yields are of primary importance to realize the LCA of algal biodiesel. Yet up to now LCA studies have demonstrated that dry extraction was too expensive in terms of energy, but at the same time there is a lack of reliable data to assess the wet extraction path.

Anaerobic digestion is mostly used to produce bioenergy from the obtained residues after lipid extraction. Energy consumption should be taken into account, and the potential meth­ane production must be more realistically assessed with existing data in order to avoid overestimation of the global energy balance. Operational parameters such as the organic load­ing rate or the hydraulic retention time should be specified, since they directly influence the energy consumption of the anaerobic process.

This book is about biofuels from microalgae. Microalgae have been used com­mercially for decades, but not for producing biofuels. Interest in algal fuels has seen a spectacular reawakening within the last 10-years. Several factors are driving the renewed quest for algal fuels: Concern about depletion of petroleum; the desire for energy independence; the need for carbon neutral renewable fuels that can be produced with­out compromising the supply of food and freshwater; and the need to prevent further deforestation. Algal fuels are not yet com­mercial and may not reach the market for long time or near-future. Nevertheless, they represent a strategic opportunity that must be persistently developed into a renewable and environmentally sustainable source of high-energy density liquid fuels.

The present book, which is the third book in the series on BIOMASS beingpublished by us, presents up-to-date state-of-art informa­tion and knowledge by the internationally recognized experts and subject peers in var­ious areas of algal biofuels. The 14 chapters of the book attempt to address many of the key issues relating to algal biofuels. Algal culture systems — open ponds as well as the closed photobioreactors — are discussed. Genetic and metabolic engineering of algae for enhanced capabilities in production of fuels are examined. Aspects of carbon fixation in industrially important microalgae are discussed. Technologies for recovering the biomass from the culture broth are assessed.

A chapter is devoted to heterotrophic produc­tion of algal oils as potential fuels. Production of fuels via fast pyrolysis of algal biomass is treated in some detail. An overview is provided of algal oils as fuels in one chapter. A chapter considers production of biohydro­gen from microalgae. Any production of algal fuels must consider the fate of the spent biomass. This is discussed in one chapter. A chapter is focused on the hydrothermal treatment of algal biomass to produce hydro­carbon fuels. Scale-up of production and com­mercialization aspects of algal fuels are examined in one chapter. A chapter discusses the life-cycle assessment of algal fuels. Changes in technology in this rapidly develop­ing field are bound to greatly diminish the environmental impact of future algal fuel production. Finally, a chapter assesses in some depth the economics of microalgal biomass production. Continuing developments will surely reduce the cost of producing algal fuels in the future.

The book would be of special interest to the post-graduate students and researchers of applied biology, biotechnology, microbiol­ogy, biochemical and chemical engineers working on algal biofuels. It is expected that the current discourse on biofuels R&D would go a long way in bringing out the exciting technological possibilities and ush­ering the readers towards the frontiers of knowledge in the area of biofuels and this book will be helpful in achieving this dis­course for algal biofuels.

We thank authors of all the articles for their cooperation and also for their prepared­ness in revising the manuscripts in a time­framed manner. We also acknowledge the help from the reviewers, who in spite of their busy professional activities, helped us by evaluating the manuscripts and gave their critical inputs to refine and improve the arti­cles. We warmly thank Dr Marinakis Kostas and Dr Anita Koch and the team of Elsevier for their cooperation and efforts in produc­ing this book.

Ashok Pandey Duu-Jong Lee Yusuf Chisti Carlos Ricardo Soccol Editors

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).

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).