Kuan-Yeow Show1 and Duu-Jong Lee2

xDepartment of Environmental Science and Engineering, Fudan University,

Shanghai, China

^Department of Chemical Engineering, National Taiwan University,

Taipei, Taiwan

4.2 INTRODUCTION

Concern has been growing over carbon emissions and diminishing energy resources related to the use of fossil fuels. To mitigate the impacts of these pressing environmental issues, extensive efforts are being made globally to explore various renewable energy sources that could replace fossil fuels. Biofuels are regarded as promising alternatives to conventional fossil fuels because they have the potential to eliminate most of the environmental problems that fossil fuels create. However, sustainable production of biofuels is hotly debated because it is perceived that biofuels produced from crops, lingo-cellulose, and food sources face various constraints in accomplishing sustainable development at the confluence of biofuel production, climate change mitigation, and economic growth. In view of the still-developing biofuel production process, biodiesel production from microalgae offers greater potential to become an inexhaustible and renewable source of energy.

Algae are a very diverse group of predominantly aquatic photosynthetic organisms that account for almost 50% of the photosynthesis taking place on Earth (Moroney and Ynalvez,

2009) . Algae have a wide range of antenna pigments to convert solar energy to chemical en­ergy via photosynthesis, giving different strains of algae their characteristic colors. Early work done with algae contributed much to what is now known about the carbon dioxide fixation pathway and the light-harvesting photosynthetic reactions. The processes of photo­synthesis in algae and terrestrial plants are very much alike. Among the three types of carbon dioxide fixation mechanisms known in photosynthetic organisms, two are found in the genus of algae (Moroney and Ynalvez, 2009). Moreover, studies indicate that carbon dioxide fixation in algae is one to two orders of magnitude higher than that of terrestrial plants
(Wang et al., 2008). Thus, algae are deemed to play a vital role in the global carbon cycle by removing excess carbon dioxide from the environment.

Cultivation of rapidly grown microalgae may acquire only 1% of land area needed for con­ventional crop-based farmlands. A microalgae production scenario estimated the use of only

121,0 hectares of open pond or 58,000 hectares of photobioreactor footprint to meet global annual gasoline requirements (Chisti, 2007). Furthermore, waste water enriched with nutri­ents such as nitrogen and/or phosphorous can be used as a growing medium for algal cul­tivation, thus negating the need for fertilizers derived from fossil-fuel energy. Additionally, uptake of nutrients by algae for biomass buildup per se is a form of treatment to the waste water in meeting effluent discharge requirements. In addition to biofuel production, culti­vated microalgae can be used as bulk commodities in pharmaceuticals, cosmetics, nutraceuticals, and functional foods (Mata et al., 2010).

Algae have been recognized as a promising biofuel resource due to their efficient conver­sion of solar energy into chemical energy. Because algae biomass is capable of producing much more oil yield per cultivation broth area than other biofuels such as corn and soy­bean crops, algal biodiesel has attracted widespread attention because of the prospect of its large-scale practical use.

Existing stages for biodiesel production from algae involve a production scheme starting with algal strain development and cultivation, followed by harvesting through separation of the algal biomass from the supporting media, and subsequent further processing such as dewatering, drying, oil extraction and fractionation (Figure 5.1). The objective of this chapter is to present a discussion of the literature review of recent developments in algae processing. The review and discussion focus on stability and separability of algae and algae-harvesting processes. Challenges of and prospects for algae harvesting are also outlined. The review aims to provide useful information to help in future development of efficient and commer­cially viable technologies for algal biodiesel production.

Chlorella is a genus of unicellular, nonmobile green microalgae first described by Beijerinck in 1890, with Chlorella vulgaris being the type species. Commonly, Chlorella cells are spherical or ellipsoidal with sizes ranging from 2 to 10 pm in diameter. They are distributed in diverse habitats such as freshwater, seawater, and soil and are free-living or symbiotic with lichens and protozoa (Gors et al., 2010). Chlorella cells reproduce themselves through asexual autospore production. Autospores are simultaneously released through rupture of the mother cell wall, with the number varying from 2 to 16. Chlorella has a thick and rigid cell wall, the structure of which may differ greatly among species.

There have been more than 100 strains of Chlorella reported in the literature. Because they lack conspicuous morphological characters, the classification of Chlorella has been problem­atic. An attempt was made to classify Chlorella species based on certain biochemical and phys­iological characters, i. e., hydrogenase, secondary carotenoids, acid and salt tolerance, lactic acid fermentation, nitrate reduction, thiamine requirement, and the GC content of DNA (Kessler, 1976). By comparing these characters, Kessler (1976) assigned 77 strains of Chlorella from the Culture Collection of Algae at Gottingen (SAG, Germany) to 12 taxa and suggested that Chlorella represents an assembly of morphologically similar species of a polyphyletic origin. Afterward, Kessler and Huss (1992) examined 58 Chlorella strains from the Culture Collection of Algae at the University of Texas at Austin using the above-mentioned biochem­ical and physiological characters and assigned them into 10 previously established species. The sugar composition of cell walls (either glucosamine or glucose and mannose) was also used as a taxonomical marker for Chlorella classification (Takeda, 1991, 1993). Using a 18S rRNA-based phylogenic approach, Huss et al. (1999) revised the Chlorella genus and consid­ered it as a polyphyletic assemblage dispersed over two classes of Chlorophyta, i. e., Chlorophyceae and Trebouxiophyceae. Only four species were suggested to be kept in the Chlorella genus: Chlorella vulgaris, Chlorella sorokiniana, Chlorella kessleri, and Chlorella lobophora. Later, Krienitz et al (2004) excluded Chlorella kessleri from the Chlorella genus and reduced the number of species to three. Here we will regard Chlorella as Chlorella sensu lato and include the data obtained from those Chlorella species that may have been excluded from the Chlorella genus by the studies mentioned.

Stirred-tank photobioreactors are the conventional reactor setup in which agitation is provided mechanically with the help of impellers or baffles by providing illumination exter­nally. CO2-enriched air is bubbled at the bottom to provide a carbon source for algae growth (Petkov, 2000; Demessie and Bekele, 2003). Protoceratium reticulatum growth studied in 2 L and 15 L stirred photobioreactors equipped with internal spin filters showed average biomass cell productivity 3.7 times higher than that of the static cultures (Camacho et al., 2011). Low surface-area-to-volume ratio, which in turn decreases light-harvesting efficiency, is the inher­ent disadvantage of this system. Low surface-area-to-volume ratio and high shear stress imposed due to mechanical agitation limits this reactor’s use in CO2 sequestration (Demessie and Bekele, 2003).

Carrageenans are composed of linear polysaccharide chains, with sulphate half-esters attached to the sugar unit. They are normally classified according to their structural charac­teristics, and there are at least 15 distinct structures.

Carrageenans are used in the food, textile, and pharmaceutical industries, where they are sought as aids to stabilize emulsions and suspensions. Most carrageenan is currently pro­duced from cell walls of Eucheuma and Kappaphycus spp. Food applications for carrageenans (usually labeled E 407) include canned foods, dessert mousses, salad dressings, bakery fill­ings, ice cream, instant desserts, and pet foods. They are also used as suspension agents and stabilizers in drugs, lotions, and medicinal creams. An illustrative medical application is treatment of bowel problems, such as diarrhea, constipation, or dysentery; they are also used to make internal poultices to control stomach ulcers (Morrissey, Kraan et al., 2001).

Heterogeneous catalysts have so far mostly been used in gasification processes, where they are reported to have a significant positive effect on low-temperature processes. In addition, during hydrothermal liquefaction some gasification is crucial, since oxygen is removed dur­ing this process. However, extensive gasification will reduce the bio-oil yield. Nickel, palla­dium, and platinum catalysts were tested during gasification of cellulose at 350°C, 25 MPa, and 10-180 min reaction time, and it was reported that mainly methane and carbon dioxide were produced over supported nickel catalysts, whereas mainly hydrogen and carbon diox­ide were produced over supported palladium and platinum catalysts. Most likely the gas is produced by direct gasification of aqueous compounds of the primary biomass degradation (Minowa and Inoue, 1999). Various other heterogeneous catalysts have been tested in hydro­thermal conversion processes; however, the main focus has been to improve gasification, not liquid yields. Examples of these catalysts are Ni/Al2O3, Ru/TiO2, and ZrO2 (Elliot et al., 1993; Elliot et al., 1994). Catalysis of gasification at conditions below 400°C was extensively reviewed by Peterson et al. (Peterson et al., 2008). In a rare study of heterogeneous catalysts at semihydrothermal conditions, Watanabe et al. (Watanabe et al., 2006) tested the effect of zirconia (ZrO2) on stearic acid (C17H35COOH) decomposition at 400°C and 25 MPa for 30 min. Zirconia has a high density of amphoteric sites on the surface, which means that it potentially promotes both acid and base-catalyzed reactions. They observed that zirconia (ZrO2) enhanced the conversion of the C17-acid, and the main products were the C16-alkene, acetic acid, and 2-Nonadecanone.

Mineral composition, C/N ratio, and growth rate of microalgae vary naturally according to environmental conditions (light and temperature), availability of nutrients, or occurrence of stress. For instance, the application of nitrogen starvation induces, for some species, the stor­age of lipids (Ketchum and Redfield, 1949). However the increase of lipid content is done to the detriment of cell division, and consequently the mass productivity is lower. Therefore, it should be highlighted that all these properties are correlated and cannot be determined on the basis of independent assumptions or sources.

As shown on Table 13.5, a large variability of productivity, lipid fraction, or nutrient re­quirement is observed among the various studies. In four publications (Lardon et al., 2009; Batan et al., 2010; Stephenson et al., 2010; Khoo et al., 2011), authors suggest to impose nitrogen deprivation on the algae. To overcome the problem of the growth-rate reduction under nutrient stresses, some authors suggest cultivating microalgae in two steps. First, microalgal biomass is cultivated in nitrogen-replete conditions in order to reach a high growth rate. Then microalgae are submitted to nitrogen deprivation to increase their lipid content.

Growth rate is known to be species dependent and strongly influenced by light and tem­perature (Falkowski and Raven, 1997). It can be strongly reduced by the stress protocol used to induce lipid accumulation by nutrient deprivation (Lacour et al., 2012). Depending on the location, cultivation system, species, and protocol, growth rate and biomass concentration can therefore vary by more than an order of magnitude. The hypotheses made in LCA studies reflect this large spectrum. In ORW, growth rates vary from 25 (Batan et al., 2010; Collet et al., 2011) to 40.6 g m-2 d-1 (Clarens et al., 2010). In PBR, productivities are much higher and vary from 270 (Jorquera et al., 2010) to 1536 g m-3 d-1 (Brentner et al., 2011). The PBR conception has a strong influence on the growth rate (Jorquera et al., 2010). Microalgae con­centrations range from 0.5 (Lardon et al., 2009) to 1.67 g L — (Stephenson et al., 2010) in an OR, and from 1.02 (Jorquera et al., 2010) to 8.3 g L-1 (Stephenson et al., 2010) in a PBR. Expected lipid contents vary broadly between authors: from 17.5% (Lardon et al., 2009) to 50% (Kadam, 2002) without nitrogen deprivation and from 25% (Khoo et al., 2011) to 50% with nitrogen deprivation (Batan et al., 2010; Stephenson et al., 2010).

To increase the recovery of cells via sedimentation, a flocculant is added to the system. Flocculation is the first step of the harvesting; this process aims to aggregate the microalgal cells and thereby increase the particle size (Grima et al., 2003). The microalgae have a negative charge on the surface to prevent cell aggregation. The loads on the surface of algae can be altered by the addition of flocculant (Harun et al., 2010).

Flocculation may be accomplished by three methods: chemical flocculation,

bioflocculation, and electroflocculation. The most common flocculants are aluminum sul­phate, aluminum chloride, and ferric chloride. The addition of sodium hydroxide raises the pH of the culture to 8-11, coagulating the cells in just a few minutes. However, the floc — culants are toxic in high concentration. Flocculants should be inexpensive, nontoxic, and ef­fective at low concentrations. Chitosan is an organic cationic polymer, a nontoxic flocculating agent that is used in wastewater treatment and in the food industry (Pires et al., 2012).

Among the various fuel categories derived from microalgae, biodiesel receives the most attention because it shares similar chemical characteristics with petrol diesel and can be directly channeled into the current transportation infrastructure without major alterations of existing technology and fuel pipelines. Oleaginous green microalgae species that have the capacity to accumulate oil in the form of triacylglycerols, or TAGs (Chisti, 2007; Sheehan et al., 1998), have been isolated and possess great potential as a feedstock for biodiesel fuels (Converti et al., 2009; Liu et al., 2008; Xu et al., 2006). However, microalgae-based biodiesel is far from being commercially feasible, because it is not economically practical at present. From a biological point of view, one of the obvious solutions is to increase oil content. Most microalgae do not accumulate large amounts of lipid during a normal growth period. Cells begin to accumulate significant amounts of storage lipids after encountering stress conditions such as light and nutrient starvation (Hu et al., 2008; Sheehan et al., 1998). Nutrient starvation, however, slows cell proliferation and therefore limits biomass and overall lipid productivity. Despite the continuous interest in and enthusiasm about microalgal oil-to-biodiesel potential, the molecular mechanisms underlying the cellular, physiological, and metabolic networks connecting to lipid and TAG biosynthesis remain largely unknown. Recent progress in transcriptomics, proteomics, metabolomics, and lipidomics studies have started to unravel the complex molecular mechanisms and regulatory networks involved in lipid and TAG biosynthesis in microalgae.

Current efforts to isolate and characterize the repertoire of genes required for lipid and TAG biosynthesis and accumulation in microalgae have focused on a model microalga: Chlamydomonas reinhardtii (Li et al., 2008; Miller et al., 2010; Msanne et al., 2012). With com­plete genome information, many enzymes required for lipid and TAG biosynthesis and me­tabolism have been identified based on in silico predictions of orthologous genes from other organisms (Riekhof et al., 2005). Similar to oilseed crops, the most common fatty acids in microalgae are 16- and 18-carbon fatty acids (Hu et al., 2008). Genome comparison and gene prediction analyses have shown that the pathways of fatty acid and lipid biosynthesis are largely conserved between plants and green algae (Riekhof et al., 2005). In plants, de novo syn­thesis of fatty acids occurs in the plastid (Ohlrogge and Browse, 1995). The synthesized fatty acids are used as building blocks for synthesis of membrane lipids and storage lipids. Acetyl CoA serves as the basic unit for fatty acid biosynthesis. It is converted to malonyl CoA by acetyl

There is limited literature on algae sedimentation in ponds without any flocculation process. Isolation of facultative oxidation pond from inflow feed to promote water clarifica­tion was investigated (Koopman et al., 1978). Operations involving fill-and-draw cycles for secondary ponds gave rise to significant removal of algae from facultative oxidation pond effluent (Benemann et al., 1980).

Similar secondary ponds were used for algae settling from high rate oxidation pond effluent (Adan and Lee, 1980; Benemann et al., 1980). Well-clarified effluent and algae slurry of up to 3% solids content were achieved at the secondary ponds attributable to algae autoflocculation, which enhanced the settling. The autoflocculation phenomenon is distinctly different from the coprecipitative autoflocculation suggested by Sukenik and Shelef (1984), as discussed earlier. The autoflocculation mechanism involved remained unclear (Eisenberg et al., 1981).

Coagulant dosing to a settling tube to promote algae sedimentation was looked into by Mohn (1980). The batched operation achieved an algal concentration of 1.5% solids content. Algae separation by sedimentation tanks or tubes is considered a simple and inexpensive process. Its concentrating reliability is low without coagulant dosing. Algae autoflocculation may be used as an inexpensive reliable algae separation method. However, the natural flocculation processes should be closely studied and well understood before it can be incor­porated for primary concentration.

The microalga used in this experiment is a nongenetially modified organism (non-GMO) adapted to a culture medium containing vinasse from an ethanol distillery. The culture was carried in open ponds (Figure 7.4) at an Ourofino Agronegcicio biofuels facility.

After being cultured, the biomass was flocculated, centrifuged, and dried. Dried algal bio­mass containing about 12% moisture was used in a fast pyrolysis system (see Figure 7.5).

The conditions of fast pyrolysis were:

Reactor temperature: 485 ± 15

Reactor pressure: 1.2 atm

Mass flow: 17kg/h

Air flow: 1.7 kg/h

The results achieved from fast pyrolysis with algal biomass are shown in Table 7.3. Elemental analyses of each fraction were carried out and are presented in Table 7.4.

To analyze the potential of fuel use, the lower heating value (LHV) was determined according to the method ABNT/NBR11956. Results are shown in Table 7.5.

Bio-oil was the fraction with higher LHV, presenting values very near some vegetable oils. For example, soy oil has an LHV of 9,500.00 kcal/kg, and babassu oil (a typical Brazilian coconut) has around 9,140 kcal/kg.

The obtained algal coal was also superior to some solid fuels, of which the LHV is around

4,0 kcal/kg.

It is important to consider the high mass density of the bio-oil: 1,230.00 kg/m3. Volumetric energetic density was then calculated as 9,927.33 kcal/L, which means the amount of energy that 1 liter of bio-oil is capable of providing. This value is 15% higher than diesel oil (8,620 kcal/L) (DalmasNeto, 2012).

The average cost of one tonne of microalgal biomass is about US$310; one liter of bio-oil produced by this technology in pilot scale is near US$1.20 per liter. This production cost will probably lower as technology scales up. Bio-oil can provide 85% of the energy that diesel oil can provide (7,922 kcal/US$ from diesel versus 6,746 kcal/US$ from bio-oil), which costs around US$1.28/L. This comparison shows the competitiveness of such bio-oil technology.

7.2 CONCLUSIONS

The technology of fast pyrolysis of algal biomass for the production of bio-oil presented very interesting results, which, combined with low cost and simplicity of operation, make this technology a potential alternative carbon-free-emission fuel process.