Centrifugation involves the application of centripetal acceleration to separate the microalgae from the culture medium (Harun et al., 2010) and is perhaps the fastest cell-recovery method based on density gradient. The centrifuge disks are easy to clean and sterilize, and centrifugation can be applied to any kind of microalga (Christenson and Sims, 2011).

Heasman et al. (2000) reported that 88-100% of centrifuged cells were viable and the col­lection efficiency was 95-100% at 13,000xg. However, the centrifuge has some disadvantages: The cells are exposed to a high gravitational force, which can alter the cell structure; the re­covery of fragile microalgae biomass requires low-speed centrifugation; the salt contained in the microalgal culture medium can cause rapid corrosion of equipment; and large-scale processes require costly equipment, such as continuous centrifuges (Pires et al., 2012).

The ability of microalgae to produce hydrogen was first reported by Gaffron and Rubin in 1942 (Gaffron and Rubin, 1942). However, the observed emission of hydrogen was transient and the amount was very minimal. In the late 1990s, Melis and co-workers demon­strated that sulfur deprivation changes cellular metabolism and allows algal culture to switch from aerobic photosynthetic growth to an anaerobic physiology state. Switching to anaerobic condition allows microalgal cultures to generate significant amounts of hydrogen for an extended period of time (Melis et al., 2000). This major breakthrough makes sustainable hydrogen production in a microalgal system a possibility. Over the years, extensive studies have been done to understand the physiology and metabolic adaptation resulting from sulfur depletion for better manipulation of biohydrogen production. Here our current understanding of hydrogen production in microalgae is highlighted and possible metabolic engineering/biotechnology strategies for improving hydrogen production are discussed.

To enhance algae settling, flat inclined plates are incorporated in a settling tank to promote solids contacting and settling along and down the plates. The slopes of plates are designed for the downgliding of the settled algal particles into a sump from which they are removed by pumping (Mohn, 1980; Shelef et al., 1984). Algae were concentrated to 1.6% solids content, and coagulant dosing was suggested if suspension of tiny algae such as Scenedesmus is fed to the system (Mohn, 1980). Operational reliability of this method was fair, and further thick­ening of algae slurry was required.

S. Venkata Mohan, M. Prathima Devi,

G. Venkata Subhash, Rashmi Chandra

Bioengineering and Environmental Center, CSIR-Indian Institute of Chemical
Technology, Hyderabad, India

7.3 INTRODUCTION

Continuous use of petroleum-derived fuels is recognized as unsustainable due to their de­pleting supplies and their contribution to the accumulation of greenhouse gases (GHG) in the environment. Biologically produced fuels have been identified as potential alternative energy sources (Posten and Schaubb, 2009; Smith et al., 2009; Rojan et al., 2011; Venkata Mohan et al.,

2011) that can mitigate GHG emissions (Hossain et al., 2008). Biofuels are being promoted as one of the most promising routes to lower CO2 emissions and to reduce the world’s depen­dency on fossil fuels (Groom et al., 2008; Smith et al., 2009). Biofuel production from renew­able sources is widely considered as one of the most sustainable alternatives to petroleum sourced fuels and a viable means for environmental and economic sustainability (Dragone et al., 2010).

Crop-based terrestrial sources of biomass face problems associated with a finite area of land available for its cultivation. In this context, algae draw much attention as an alter­native source of biomass that is capable of generating fuel. Compared to crop-based coun­terparts, algae have rapid growth rates. It is estimated that algae could yield 61,000 liters per hectare (L/ha), compared with 200 L/ha to 450 L/ha from crops such as soya and ca­nola (Duan and Savage, 2010). Algae are a known rapidly growing species of which the carbon-fixing rates are much higher than those of terrestrial plants. Microalgae commonly double their biomass within 24 hours (h), and this duration during the exponential growth phase can be as short as 3.5 h (Harrison et al., 2012; Chisti 2007). The prominence of algae — based biofuels evolved due to their domestic origin, carbon neutrality, renewability, abun­dant availability, higher combustion efficiency, and higher biodegradability (Zhang et al., 2003). Different algal species showed varied lipid content (Prymnesium paryum, 22-38%;

Chlamydomonas rheinhardii, 21%; Chlorella vulgaris, 22%; Spirogyra sp., 11-21%; Scenedesmus obliquus, 12-12%; Scenedesmus dimorphous, 16.40%; Porphyridium cruentum, 4-14%; Synchoccus sp., 11%; Dunaliella bioculata, 8%; Tetraselmis maculate, 3%; based on dry biomass) (Becker, 1994; 2004).

Photosynthesis has been recognized as an efficient carbon sequestration mechanism. Microalgae can sequester atmospheric CO2 (Chisti, 2007) and utilize carbon as well as in­organic nutrients present in wastewater for their growth and survivability (Venkata Mohan et al., 2011). During photosynthesis, microalgae capture atmospheric CO2, resulting in the synthesis of carbohydrates. Creating stress on microalgae at this stage causes the photosynthetic mechanism to switch from enhancing the biomass to accu­mulating lipids. The intracellular lipid granules stored under stress conditions act as precursors for fatty acid biosynthesis. The triglyceride composition of algae upon transesterification with an alcohol can produce algae-derived biodiesel (alkyl esters). Depending on the species, growing conditions, and growth stages, microalgae have been shown to produce various types of lipids including triacylglycerides, phospholipids, gly — colipids, and betaine lipids (Greenwell et al., 2010). Microalgae-derived lipids and biomass can be converted into alcohols, methyl esters, and alkanes for use in spark-ignited engines, compression ignition engines, and aircraft gas turbine engines (Harrison et al., 2012). Under specific cultivation conditions, algal oil content can exceed 50% by weight of dry biomass (Chisti, 2007). According to an estimate, the productivity of algae-derived biofuels is predicted to be on the order of 5,000 gallons/acre/year, which is approximately two or­ders of magnitude greater than the yield from terrestrial oil seed crops such as soybeans (Demirbas, 2007; Weyer et al., 2009).

Biofixation/sequestration of CO2 using photosynthetic microalgae is one potential op­tion for harnessing renewable energy. Cultivation of algae for biodiesel production is con­sidered more beneficial to the environment than the cultivation of oil crops (Chisti, 2007) because the productivity of algae-derived oils is much higher than the best oil-producing crops (Abou-Shanab et al., 2010). Compared to fossil-driven fuels, microalgae-based biofuels are renewable, biodegradable, and eco-friendly (Ma and Hanna, 1999; Knothe, 2006; Vicente et al., 2010). The cultivation of algae doesn’t require arable land, since they can be grown in artificial ponds, on land that’s unsuitable for agriculture, on surfaces of lakes or coastal waterways, or in vats on wasteland (Duan and Savage, 2010). Algal-based fuel addresses the major constraints posed by the first — and second-generation biofuels due to its fast growing nature and capability to produce several times higher biomass com­pared to terrestrial crops and trees, requires low and marginal land and other resources, produces higher lipid and carbohydrate, and so on (Singh et al., 2011). Production of biofuels from microalgae is gaining acceptance because of its economic feasibility and en­vironmental sustainability compared to agro-based fuels. Microalgae-derived biofuels have the potential for scalability (Harrison et al., 2012). Algae-derived biodiesel is currently being promoted as a third-generation biofuel feedstock since algae doesn’t compete with food crops and can be cultivated on nonarable land (Dragone et al., 2010). In writing this chapter, a comprehensive attempt was made to summarize the basic and applied aspects of algal-based fuel by synthesizing the contemporary literature in conjunction with recent developments.

The conversion of microalgae oil to biodiesel by direct transesterification involves both extraction and esterification in a single step (Sathish and Sims, 2012; Ehimen et al., 2010). The algae biomass can be effectively converted to fatty acid methyl esters through this process in relatively less time. Minimization of solvents and requirement of less time for reactions are the advantages of this method; the lipid productivity and success rate of the reactions are the associated drawbacks.

8.7.1 Acid-Catalyzed Transesterification

The acid-catalyzed transesterification process involves an acid catalyst (H2SO4/HQ) to undergo the reaction. These reactions are usually performed at high alcohol-to-oil-molar ra­tios, low to moderate temperatures and pressures, and high acid-catalyst concentrations (Zhang et al., 2003). Compared to base catalysts, acid catalysts are less susceptible to the pres­ence of free fatty acids in the source feedstock (Helwani et al., 2009), but the reaction rates of
converting triglycerides to methyl esters are too slow (Gerpen, 2005). Repeated application of catalyst in the reactions increases the acid value of the microalgae oil.

Unlike chlorophylls and carotenoids, phycobiliproteins are water-soluble and form particles (phycobilisomes) on the surface of thylakoids rather than being embedded in the membranes. These proteins are major photosynthetic accessory pigments in algae and include phycoerythrin, phycocyanin, allophycocyanin, and phycoerythrocyanin (Jian-Feng, Guang-Ce et al., 2006).

Phycobiliproteins consist of pigmented phycobilins, i. e., linear tetrapyrroles. Various combinations of the two major phycobilins—phycoerythrobilin (red) and phycocyanobilin (blue)—can absorb at distinct spectral regions (Lobban and Harrison, 1994). Within phycobilisomes, phycobiliproteins play an important role in the photosynthetic process of at least three families of algae: Rhodophyta, Cyanophyta, and Cyptophyta (Chronakis, Galatanu et al., 2000; Aneiros and Garateix, 2004). The additional photosynthetic pigments make light harvesting possible in deep waters because surface light wavelengths for some colors are almost completely absorbed below 10 m (Voet, Voet et al., 2008).

The aforementioned proteins have been used as natural colorants for food and cosmetic applications, e. g., chewing gum, ice sherbets and gellies, and dairy products, in addition to lipsticks and eyeliners (Bermejo Roman, Alvarez-Pez et al., 2002; Sekar and Chandramohan, 2008). Several phycobiliproteins have been shown to exhibit antioxidant, anti-inflammatory, neuroprotective, hypocholesterolemic, hepatoprotective, antiviral, antitumoral, liver-protecting, serum lipid-reducing, and lipase-inhibiting activities (Sekar and Chandramohan, 2008). Therefore, such health products as tablets, capsules, or powders that include phycocyanin have successfully reached the market in recent times (Guil-Guerrero, Navarro-Juarez et al., 2004). This type of pigment can be recovered by several techniques, e. g., solvent extraction and pressurized liquid extraction as well as expanded bed absorption chromatography, as covered by Liam et al. (Liam, Anika et al., 2012).

To commercialize algal biofuels, the first challenge is the mass production of algal biomass with minimal energy input and in a cost-effective manner. Phototrophic cultivation appears to be the preferred method to cultivate algae because sunlight is abundantly available at no cost. Apart from that, phototrophic algae are able to capture CO2 from flue gases and could potentially act as a superior carbon sink, offering an added advantage to this cultivation method. However, this method has its limitations, especially in temperate countries where suitable sunlight intensity is not always available throughout the year (Lam and Lee, 2012). The open pond system and the closed photobioreactor are among the cultivation systems that are suitable for growing phototrophic algae. An ideal cultivation system should meet the fol­lowing requirements: (1) has a large effective illumination area, (2) utilizes optimal gas-liquid transfer, (3) is simple to operate, (4) maintains a low contamination level, (5) has low capital and operating costs, and (6) utilizes a minimal amount of land (Xu et al., 2009). Unfortunately, this ideal cultivation system is yet to be realized, even with intensive research and field trials.

The following section details the basic design of the open and closed photobioreactors, including their advantages, limitations, and factors to consider before attempting to scale up both cultivation systems.

13.6.1 Perimeter and Functional Units

The lack of inclusion of biofuel combustion from the perimeter of the study can facilitate the comparison between different technologies or energy production pathways, but it ham­pers the assessment of the real carbon balance; indeed, some of the carbon atoms of the methylester stem from methanol, which is usually produced from fossil fuel (Stephenson et al., 2010). Moreover, it ignores environmental impacts from combustion (such as photo­chemical oxidation and particulate matter formation). Finally, all engines do not have the same efficiency, and hence a fair comparison should be based on the available work produced by the use of the fuel rather than on the chemical property of the fuel only.

To harmonize LCA results and provide a better basis for comparisons, the energy content of intermediate products (raw algae, oil, oil extraction residues, and methylester) should be systematically provided and justified. We also recommend using the LHV instead of the HHV; indeed, in most cases, biofuel will be used in engines (internal combustion engines or turbines) that are unable to use the energy stored in the water vapor resulting from fuel combustion.

As shown in the preceding section, the choice of using allocation or substitution to handle the multifunctionality of processes has a strong influence on the results. Even though the sys­tem expansion is a priori preferable, it can lead to an increase in the overall uncertainty when performance of substituted processes are little known (performance of anaerobic digestion of oilcakes) or if the validity of the substitution is questionable (use of oilcake extraction as an­imal food, for instance).

Since 1940, the most widely used plastics have been polyethylene (PE), polypropylene (PP), polystyrene (PS), poly(ethylene terephthalate) (PET), and poly(vinyl chloride) (PVC). Despite advances, plastics processing and manufacturing generate two major problems: the use of nonrenewable resources to obtain their raw materials and large quantities of waste generated for disposal.

Biodegradable plastics degrade completely within three to six months when attacked by microorganisms, depending on the environmental conditions. The polyhydroxyalkanoates (PHAs) are natural polyesters consisting of units of hydroxyalkanoic acids with similar prop­erties to petrochemical plastics (Jau et al., 2005).

The polyhydroxyalkanoates are produced as a reserve of carbon and energy accumulated within the cells of various microorganisms such as microalgae. Among the PHAs, polyhydroxybutyrate (PHB) and its copolymer polyhydroxybutyrate-co-valerate (PHB — HV) are synthesized by cyanobacteria when exposed to specific conditions of cultivation (Sharma et al., 2007).

The degradation rate of PHB and PHB-HV depends on many factors, some related to the environment, such as temperature, moisture, pH, and nutrient supply, and others related to the biopolymer itself, such as composition, crystallinity, additives, and surface area. Due to its physical and chemical properties, PHB is easily processed in equipment commonly used for polyolefins and synthetic plastics (Khanna and Srivastava, 2005).

The first photosynthetic microbe to be isolated and grown in pure culture was the fresh­water microalga Chlorella vulgaris. It is a spherical unicellular eukaryotic green algae that presents a thick cell wall (100-200 nm) as its main characteristic. This cell wall provides mechanical and chemical protection, and its relation to heavy metals resistance is reported, which explains why C. vulgaris is one of the most used microorganisms for waste treatment.

The uptake of carbon by C. vulgaris cells is done through the enzyme carbonic anhydrase, which catalyzes the hydration of CO2 to form HCO3 and a proton. Hirata and collaborators (1996) studied carbon dioxide fixation by this microalga, which showed important variations comparing cultivation under fluorescent lamps and sunlight. In the first case the estimated rate of carbon dioxide fixation was 865 mg CO2 L-1 d-1; in a sunlight regimen the estimated rate achieved 31.8 mg CO2 L-1 d-1. Winajarko et al. (2008) achieved a transferred rate of

441.6 g CO2 L-1 d-1 under the same cultivation conditions as Hirata et al. (1996). According to Sydney et al. (2011), in experiments using classic synthetic media and a 12-h light/dark regimen, C. vulgaris biofixation rate of carbon dioxide is near 250 mg L-1 day-1.

Carbon fixation by Chlorella vulgaris is variable and depends, among other factors, on the concentration of CO2 in the gaseous source. Yun et al (1997) cultivated C. vulgaris in 15% of carbon dioxide and achieved a fixation of 624 mg L-1 day-1; Scragg et al. (2002) achieved a fixation of 75 mg L-1 day-1 under CO2 concentration of 0.03%. In the same study, Scragg tested a medium with low nitrogen and the fixation rate was 45 mg L-1 day-1, suggesting that nitrogen also influences carbon uptake rate.

Some studies (Chinassamy et al., 2009; Morais and Costa, 2007) indicate that the best concentration of CO2 in the gas supplied to C. vulgaris growth is about 6%.