To enhance the economics of microalgae-based biofuels, utilization of every ingredient of the raw biomass is important (Georgianna and Mayfield, 2012; Sheehan et al., 1998). Whereas the majority of fuels derived from microalgae have been focused on storage oils, the extracted oil accounts for only 37.9% of the energy and 27.4% of the initial fixed carbon (Lardon et al.,

2009) . The remaining carbon is stored in the leftover oil cakes composed of abundant proteins and carbohydrates. Hence, recycling these nutrient elements may help increase biomass margins of microalgae-based fuels (Lardon et al., 2009). Recycling algal waste by anaerobic digestion has been proposed to support the microalgae production process (Ras et al., 2011; Zamalloa et al., 2012).

Several innovative metabolic engineering strategies have been proposed recently to reduce the energy debt and increase the margins of microalgae-based fuels. One of the approaches is to establish an integrated system that takes advantage of the amenable genetic modification capability of the Escherichia coli (E. coli) system. Although microalgae can grow photosynthet­ically to accumulate biomass for biodiesel purposes, the leftover paste can be utilized for alcohol-fuel production by feeding it into an engineered bacterial system. Huo et al. accom­plished this by genetically engineering an E. coli strain that is capable of converting the back­bone and side chains of amino acids in pretreated biomass into two-, four — and five-carbon alcohol fuels, ammonia, and other chemicals (Huo et al., 2011). In a small-scale experiment, the authors successfully converted hydrolyzed microalgal protein biomass into alcohol fuels. This demonstration supports the potential of using microalgal biomass as a feedstock for protein-based biorefinaries.

Jin Liu1, Zheng Sun2, Feng Chen3

institute of Marine and Environmental Technology, University of Maryland Center for
Environmental Science, Baltimore, MD, USA
2School of Energy and Environment, City University of Hong Kong, China
3Institute for Food & Bioresource Engineering, College of Engineering,

Peking University, Beijing, China

6.1 INTRODUCTION

Petroleum fuels are recognized as unsustainable due to their depleting supplies and re­lease of greenhouse gas (Chisti, 2008). Renewable biofuels are promising alternatives to pe­troleum and have attracted unprecedentedly increasing attention in recent years (Hu et al.,

2008) . Compared with traditional fuels, the carbon-neutral biodiesel releases less gaseous pol­lutants and is considered environmentally beneficial. Currently biodiesel is mainly produced from vegetable oils, animal fats, and waste cooking oils. Plant oil-derived biodiesel, however, cannot realistically meet the existing need for transport fuels, because immense amounts of arable land have to be occupied to cultivate oil crops, causing a fuel-versus-food conflict (Chisti, 2007). Because of their fast growth and lipid abundance, microalgae have been con­sidered the promising alternative feedstock for biofuel production, and their potential has been widely reported by many researchers in recent years (Chisti, 2007, 2008; Hu et al., 2008; Mata et al., 2010; Liu et al., 2011a).

Mass cultivation of microalgae started almost concurrently in United States, Germany, and Japan in the late 1940s (Burlew, 1964). From then on, the mass culture of algae became one of the hottest topics in algal biotechnology, and increasingly improved culture systems have been developed (Hu et al., 1996; Lin, 2005; Chisti, 2007; Masojidek et al., 2011). Nowadays the most common procedure for mass culture is autotrophic growth in open ponds, where the microalgae are cultured under conditions identical to the external environment. Circular ponds are the most common device for the large-scale commercial production of Chlorella (Lin, 2005). Circular ponds were first built in Japan and then introduced to Taiwan and now are widely adopted in Asia. The size of circular ponds may range from 30 to 50 m in diameter, and a rotating agitator provides culture mixing.

Raceway ponds are another popular open culture device for mass culture of Chlorella. They are made from poured concrete or simply dug into the earth covered with a plastic liner and are either set as individual units or arranged as a meandering channel assembled by multiple individual raceways. The culture usually is 20-30 cm in depth and circulated by a motorized paddlewheel.

Although the open pond systems cost less to build and operate and are more durable, with a large production capacity compared to a more sophisticated closed photobioreactor (PBR) design, they have substantial intrinsic disadvantages, including difficulties in managing cul­ture temperature, insufficiency of CO2 delivery, poor light availability on a per-cell basis, rapid water loss due to evaporation, susceptibility to microbial contamination, poor growth, low cell concentration, and consequently high cost for biomass harvest.

To overcome the inherent limitations associated with open pond systems, closed PBRs of various geometries and configurations were adopted for mass cultivation of microalgae. A popular PBR is a tubular design that is made of clear transparent tubes of a few centimeters in diameter and arranged in various configurations, e. g., a serpentine shape placed above the ground, multiple tubes running in parallel and connected by a manifold structure, a-type cross tubes at an angle with horizontal, or coiled tubes helically around a supporting frame (Lee et al., 1995; Borowitzka, 1999).

Flat plate PBR is another type of PBR design that may be arranged either vertically or in­clined to the ground (Tredici et al., 1991; Hu et al., 1996,1998; Zhang and Richmond, 2003). Although capable of producing much higher cell densities than open ponds, they proved dif­ficult to scale up, and the capital in infrastructure and continuous maintenance may be high. In addition, the light limitation and oxygen accumulation associated with the buildup of cells in PBRs are problematic issues that remain to be resolved.

Due to the significant characteristics such as fast growth, ultrahigh cell density, and high oil productivity associated with heterotrophic algae, heterotrophic production of algal oils has received substantially increasing interest and the scale-up production for possible com­mercialization is sought, though it may be regarded as less economically viable than using autotrophic growing algal cultures for producing lipid-based biofuels. This chapter provides an overview of the current status of using heterotrophic algae—in particular, Chlorella—for oil production. The path forward for further expansion of the heterotrophic production of algal oils with respect to both challenges and opportunities is also discussed.

Cultivation of algae in open ponds mimics the natural method of growing algae (Pearson, 1996; Chisti, 2007). Open ponds can be categorized into natural waters (lakes, lagoons, ponds, etc.) and artificial ponds or containers. The most commonly used systems include shallow ponds (large in size), raceway ponds, tanks, and circular ponds. Raceway ponds generally consist of an oval-shaped shallow pond lined with PVC, cement, or clay, having an area of 1-200 ha (Andersen, 2005). Ponds are divided by a series of baffles, and water is moved through the ponds in order to promote mixing of nutrients and uniform algae growth. These ponds are usually constructed in shallow dimensions as the algae need to be exposed to sunlight, and sunlight can only penetrate the water up to a certain limited depth (Chisti, 2007). The ponds are operated in a continuous mode, with CO2 and nutrients being constantly fed to the pond while the algae-containing water is removed at the other end. Large open-pond cultivation for mass algal production of single-cell protein, health food, and beta-carotene is one of the oldest industrial systems since the 1950s (Chisti, 2007; Perez-Garcia et al., 2011).

Cultivation of microalgae in open ponds presents relatively low construction and operat­ing costs, which invariably result in low production costs (Stephenson et al., 2010; Chen, 1996; Tredici, 2004). Large ponds can be constructed on degraded and nonagricultural lands that avoid the use of high-value lands and crop-producing areas (Chen, 1996; Tredici, 2004). On the contrary, open pond cultivation inherits some drawbacks such as poor light diffusion, losses due to evaporation, CO2 diffusion from the atmosphere, and the requirement of large areas of land (Harun et al., 2010; Perez-Garcia et al., 2011). Furthermore, contamination by predators and other fast-growing heterotrophs restricts the commercial production of algae in open-air pond/culture systems. Not-so-efficient mixing in open cultivation permits poor mass transfer rates, resulting in low biomass productivity (Pulz, 2001; Harun et al., 2010). Uncontrolled environments in and around the pond pose a multitude of problems that can directly or indirectly stunt algae growth (Mata et al., 2010). Uneven light intensity and distribution within the pond (Kazamia et al., 2012) and uncontrolled pond temperature also have a significant influence on the algal biomass productivity.

Algae can produce, but they can also behave as material for production of several biofuels. The main possibilities will be scrutinized below, focusing on reuse of spent biomass for complementary production of secondary biofuel.

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).

12 1 Pierre Collet1, Daniele Spinelli2, Laurent Lardon1, Arnaud

Helias1’3 , Jean-Philippe Steyer1, Olivier Bernard4

1INRA UR0050, Laboratoire de Biotechnologie de l’Environnement,

Avenue des Etangs, Narbonne, France
2Department of Chemistry and Center for Complex System Investigation,
University of Siena, Siena, Italy
3Montpellier SupAgro, Montpellier, France
4INRIA BIOCORE, Sophia Antipolis Cedex, France

13.1 INTRODUCTION

Environmental impacts and depletion of fossil energies have promoted the development of alternative and renewable sources of energy. Nonetheless, it is clear now that the replacement of current fossil energy will require both the development of new strategies to reduce our global energy consumption and the development of a panel of renewable energy sources. Re­newable energy can be extracted from solar, wind, or geothermal energy. However, these en­ergy forms are globally hard to store and hence cannot yet replace our consumption of fossil fuel for some important functions, such as powering cars and planes.

So far, several paths have been explored to produce fuels from renewable sources, the most developed strategies leading to the production of so-called first — and second-generation biofuels. First-generation biofuels are based on fuel production (ethanol or methylester) from a currently cultivated and harvested biomass (e. g., corn, rapeseed). Second-generation biofuels correspond to the development of new energy production pathways from usual feed­stock not reclaimed by food production (e. g., straw or wood). The development of first- generation biofuels has been criticized—first, because of the direct competition they create with food crops in a context where food security is a raising concern, and second, because of their actual poor environmental performance. Indeed, inputs to production (e. g., fertilizer or pesticides), feedstock culture, harvest, and transformation imply fuel consumption and lead to new pollutant emissions (Borjesson and Tufvesson, 2011), especially emissions of greenhouse gases to the environment (Searchinger et al., 2008). Consequently, energy and en­vironmental benefits of these biofuels are limited. Second-generation biofuels improve envi­ronmental performance but are not free of criticisms.

These observations advocate for the necessity of systematically assessing new energy pro­duction paths with a life-cycle assessment (LCA) perspective, which means, first, the adoption of a cradle-to-grave perspective, that is, looking at resource depletion, energy consumption, and substance emissions of all the processes required to achieve the production and use of the fuel, and second, the assessment of several environmental impacts, not only global warming potential or net energy production. LCA is an ISO method developed with this aim: It allows the detection of pollution transfer from one step to another or from one kind of environmental impact to another. The European Directive on Renewable Energy (European Union, 2009), adopted in 2009, embraces LCA as a reference method to assess environmental impacts of biofuel and to meet greenhouse gas reduction objectives of 50% in 2017.

Third-generation biofuels correspond to the development of bioenergy productions based on new feedstock reputed to have lower land competition. Microalgae belong to this third category. Their very high actual photosynthetic yield and their ability to accumulate lipids, or, for some species, starch, added to the possibility that they can be cultivated in controlled environments, promise the potential of biofuel that has a low competition with food crops (Chisti, 2007) and limited environmental impacts. In addition, the ability to use CO2 directly from industrial emissions as a source of carbon for the growth of microalgae is a promising feature for flue-gas mitigation (Huntley and Redalje, 2007; Chisti, 2007). However, this promise should be challenged. So far, microalgae industrial production has been developed only for the production of high-value molecules (such as beta-carotenes) or dietary supplements (Spirulina or Chlorella can be found as pills in health shops); hence, energy or environmental performance has never been a concern. Moreover, the scale of the existing facilities is far smaller than that required for fuel pro­duction (at least several hundred ha). It is necessary to assess the expected environmental performance of these potential production systems in order to detect technological bottle­necks and to determine which processes should be optimized in priority. This approach is now necessary to design a sound, energetically efficient, and environmentally friendly bio­fuel production system.

Since the new focus of international scientific and economic communities on microalgae — based biofuel, many environmental, energy, or economical assessments have been published, with different final energy carriers or different production assumptions. Here we propose to review a set of publications, all of them published in peer-reviewed scientific journals, using the LCA method to assess the environmental impacts linked to microalgae-based biofuel. The lack of real industrial facilities dedicated to energy production from microalgae imposes the use of models and extrapolations to describe the production systems. In addition, system frontiers and coproduct management differ among the studies. Altogether, this leads to di­vergences between publication results. This review aims to identify and explain this variabil­ity and then to propose guidelines to improve future LCAs of algal-based bioenergy production systems. This work is a mirror of this diversity and underlines the difficulty in comparing different studies without common assumptions.

The greatest issue in agriculture nowadays is the availability of chemical fertilizers at af­fordable costs. Nitrogen fixation has been acknowledged as the limiting factor in food pro­duction. The concept of using cyanobacteria to fix nitrogen is based on the ability of these microalgae to grow in soil.

The microalgae Nostoc, Anabaena, Oscillatoria, Cylindrospermun, and Mastigocladus Tolypothrix form heterocysts and can fix nitrogen aerobically. Nonheterocyst-forming fila­mentous microalgae, such as Oscillatoria and Phormidium, can fix nitrogen in the absence of oxygen and in the presence of nitrogen and carbon dioxide. Filamentous forms without heterocysts, such as Trichodesmium, may fix nitrogen aerobically (Richmond, 1990).

The heterocysts, which are specialized in aerobic nitrogen fixation, are the site of the en­zyme nitrogenase, which catalyzes the conversion of nitrogen into ammonia. Nitrogen-fixing cyanobacteria were isolated in soils from various cities in South Asia, India, and Africa. In that study, 33% of 2,213 soil samples collected in India contained cyanobacteria. Microalgae such as Nostoc, Anabaena, Calothrix, Aulosira, and Plectonema were found in soils in India, while Halosiphon, Scytonema and Cylindrospermum were observed in the other regions (Richmond, 1990).

1.2 CONCLUSION

Open ponds are the most widely used reactors in the world for large-scale microalgal cul­tures. This is due to the low construction cost, low power demand, appropriate scale-up, and their easy cleaning process compared to closed photobioreactors. The cultures that are grown in open ponds can be protected from adverse environmental conditions (rainfall, tempera­ture, and luminosity) through the use of a greenhouse. Microalgae that grow in extreme conditions, such as an alkaline medium and high salinity, should be adopted in order to achieve axenic cultures. The obtained microalgal biomass can be used in the production of food, drugs, biopigments, biopolymers, biofuels, and biofertilizers.

Spirulina are multicellular ilamentous cyanobacteria actually belonging to two separate genera: Spirulina and Arthrospira. These encompass about 15 species (Habib et al., 2008). This microorganism grows in water, reproduces by binary fission, and can be harvested and processed easily, having significantly high macro — and micronutrient contents. Their main photosynthetic pigments are chlorophyll and phycocyanin. The helical shape of the filaments (or trichomes) is characteristic of the genus and is maintained only in a liquid environment or culture medium.

Spirulina is found in soil, marshes, freshwater, brackish water, seawater, and thermal springs. Alkaline, saline water (>30 g/L) with high pH (8.5-11.0) favors good production of Spirulina, especially where there is a high level of solar radiation. It predominates in higher pH and water conductivity. Like most cyanobacteria, Spirulina is an obligate photoautotroph,

i. e., it cannot grow in the dark on media containing only organic carbon compounds. It reduces carbon dioxide in the light and assimilates mainly nitrates.

Spirulina contains unusually high amounts of protein, between 55% and 70% by dry weight, depending on the source. It has a high amount of polyunsaturated fatty acids (PUFAs), 30% of its 5-6% total lipids, and is a good source of vitamins (B1, B2, B3, B6, B9, B12, C, D, E). Spirulina is a rich source of potassium and also contains calcium, chromium, copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc. These bacteria also contain chlorophyll a and carotenoids.

The optimum pH of the Spirulina sp. culture is between 8.5 and 9.5 (Watanabe et al., 1995). Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO — transport. In experiments conducted by Morais and Costa (2007), carbon fixation in terms of biomass by Spirulina platensis was estimated in 413 mg L-1 d-1, near those achieved by Sydney et al. (2011).

Carlos Jose DalmasNeto1, Eduardo Bittencourt Sydney2,
Ricardo Assmann1, DolivarCoraucci Neto1,

Carlos Ricardo Soccol2

xOurofino Agronegocio, Rodovia Anhanguera SP 330, Km 298 Distrito Industrial,

Cravinhos, SP, Brazil

2Department of Bioprocess Engineering and Biotechnology,

Federal University of Parana, Curitiba-Pr, Brazil

7.1 INTRODUCTION

In recent years microalgae are gaining importance mainly due to their potential for fuel production with zero carbon emissions. In the actual context, algal fuel is economically unfeasible compared to petroleum-derived fuel (which costs around US$0.55/L to U. S. con­sumers). To successfully make the transition from fossil fuels to biofuels, it is necessary to achieve the same or better quality (chemical and physical characteristics) for at least the same price. At this point, for most of the world, economics have greater influence than the eco­friendly characteristics (renewable sources and less polluting gas emissions) offered by biofuels.

The main reason for this economical limitation of biofuels manufactured from algae is the high costs of culture media and downstream processes (extraction, purification, and transformation) on an industrial scale. To make algal oil technologies economically feasible, these steps might be improved. In terms of culture media, it is in vogue to use wastewater as a partial or complete source of nutrients (carbon dioxide, nitrogen, phosphorous, potassium, magnesium, and some micronutrients) for algal growth as an alternative to reduce cultivation costs, whereas in terms of oil recuperation and transformation fast pyrolysis is a cheap alternative. This chapter describes a patented technology for biofuel production through fast pyrolysis from lipid-rich microalgae.

Microalgae are composed of single cells surrounded by an individual cell wall, which in­cludes "unusual" lipid classes and fatty acids that differ from those in higher animals and plants (Guschina and Harwood, 2006). For extraction of lipids from microalgae, regular extraction methods may not be applicable (Eline et al., 2012). Extracting and purifying oil from algae is considered challenging due to its energy — and economically intensive nature (Fajardo et al., 2007; Lee et al., 2010; Mercer and Armenta, 2011).

8.6.2.1 Solvent Extraction

The existing procedures for the extraction of lipids from source material usually involve selective solvent extraction, and the starting material may be subjected to drying prior to ex­traction (Lee et al., 2010). Lipids are soluble in organic solvents but sparingly soluble or in­soluble in water. Solubility of lipids is an important criterion for their extraction and typically depends on the type of lipid present and the proportion of nonpolar lipids (princi­pally triacylglycerols) and polar lipids (mainly phospholipids and glycolipids) in the sample (Huang et al., 2010). Several solvent systems are used, depending on the type of sample and its components. The solvents of choice are usually hexane in the case of Soxhlet and Goldfish methods (Additions and Revisions, 2002); chloroform/methanol or chloroform/methanol/ water in the case of the Folch Method (Folch and Sloane-Stanley, 1957); or modified Bligh and Dyer Procedure (Bligh and Dyer, 1959). This method is best suited to extract nonpolar lipids because polar lipids are scarcely soluble in nonpolar solvents.