When a fluid is subjected to temperatures and pressures above its critical temperature and pressure, it becomes a supercritical fluid. Supercritical fluid extraction (SFE) is the process of extracting oil from oil-containing materials using a supercritical fluid as the extraction solvent. The advantage of supercritical fluids used in oil extraction is their increased solvating power (Mercer and Armenta, 2011). Factors to consider when selecting an SFE solvent include that the solvent is nonflammable, nontoxic, has low critical parameters, good solvating properties, is easily separated from prod­uct, and is environmentally friendly and inexpensive. The added advantages of SFE over conventional solvent extraction are that it provides simple and flexible process control of temperature, shorter extraction times, low cost, and solvent-free product. The SFE consists of an SFE solvent tank, solvent and feed pumps, a high-pressure pump, extraction vessels, and restrictor and absorbent vessels. Carbon dioxide (CO2) is widely used as a solvent in SFE due to its moderate critical temperature (31.1°C) and pressure (72.9 atm) (Cooney et al., 2009). In this method, CO2 is used as the extracting solvent when it is in a supercritical state (i. e., it has both gas and liquid properties). The supercritical state of CO2 can be achieved by liquefying CO2 under higher pressure and heating to a particular temperature. The important operating parameters considered for optimizing the extraction efficiency of this method are operating temperature and pressure, quantity of CO2 supplied, feed particle size, and residence time. Dried algae paste must be used for supercritical extraction; this helps in increasing the contact time between the SFE solvent and the algae paste. CO2 acts as a gas in air at ambient temperature, and can be removed after the

Comparison of different oil extraction methods

TABLE 7.2 S. Mechanical Solvent Supercritical Fluid No. Parameters Pressing Extraction Extraction Ref. 1 Algae form Dry Dry Dry Sahena, 2009; Singh and Sai, 2011; de Boer et al., 2012 2 Pretreatment to algae biomass for cell rupture Required to improve lipid extraction efficiency Required to improve lipid extraction efficiency Not required Cooney et al., 2009; Sahena et al., 2009; Mercer and Armenta, 2011; de Boer et al., 2012 3 Solvent used No solvent Hexane Supercritical CO2 Popoola and Yangomodou, 2006; Cooney et al., 2009; Demirbas, 2009 4 Working fluid Not applicable Toxic Nontoxic Mercer and Armenta, 2011 5 Oil recovery (%) 70-75 96 100 Demirbas, 2009; Popoola and Yangomodou, 2006; Demirbas and Demirbas, 2010 6 Purity of oil NA Both polar and nonpolar and colors Only nonpolar and colors Cooney et al., 2009; Mercer and Armenta, 2011 7 Energy consumption Low High High Macias-Sanchez et al., 2005; Cooney et al., 2009; Mercer and Armenta, 2011; de Boer et al, 2012 Capital investment Low High High Mercer and Armenta, 2011 8 Recycling unit Nil Necessary for reuse Necessary for reuse — 9 Scaling up Possible Possible Difficult at this time Pawliszyn, 1993; Macias-Sanchez et al., 2005; Mercer and Armenta, 2011

extraction and reused again for further extractions. A comparison of three different oil extraction methods used for lipid recovery is provided in Table 7.2.

7.2 CONCLUSION

Microalgae are becoming more attractive feedstocks for biodiesel production as higher oil-yielding algae have the potential to replace conventional biodiesel feedstocks. The viability of microalgae oil-based biodiesel production primarily depends on the identification of appropriate higher lipid producing algal strains. From preliminary studies on lipid analysis for identifying algae, important fuel properties of the algal oil can be predicted and compared with biodiesel standards. The simplest method of assessing the fuel quality of biodiesel is predicting its fuel properties based on the fatty acid composition of algal oil, thereby allowing us to ascertain the suit­ability of selected algae strains for biodiesel production. The economical/technical viability of microalgal oil-based biodiesel production depends on implementation of the suitable technologies used in the downstream processing of microalgae.

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Apart from the key algal compound groupings, mentioned above, there are new market sectors and applications emerging in algal biotechnology.

10.2.4.1 Cosmetic Extracts

Marine microalgae contribute to a range of extracts rich in proteins, vitamins, and minerals, which are incorporated as active ingredients into a number of cosmetic products (Kim et al., 2008). In addition to carotenoids, phycobiliproteins, and PUFAs, microalgae produce a number of other compounds (that exhibit a range of benefits) appealing to cosmetic formulators (Table 10.8).

These compounds prevent blemishes, repair damaged skin, aid in the treatment of seborrhoea (greasy skin caused by excess sebum), and inhibit the inflammation process (Kim et al., 2008). They are formulated into face and skin care products,

TABLE 10.8

Microalgal Compounds and Their Cosmeceutical Properties

Cosmeceutical Properties

Skin protection against UV radiation Antioxidant

Protection against UV irradiation or oxidative damage

Prevention of light-induced pathologies of the human skin and eyes

Prevention of degenerative disorders (atherosclerosis, cardiovascular disease, and cancer)

Antioxidative action

Protection against UV irradiation

Antioxidant

Emmolient

Blood stimulant

Diuretic

Moisturizing activities

Source: Adapted from Kim et al. (2008).

such as anti-aging creams and moisturizers, sun protection products, hair care products, refresherant or regenerant care products, emollients, and anti-irritant skin peels (Spolaore et al., 2006; Carlsson et al., 2007). Arthrospira (Spirulina) and Chlorella sp. are the two main genera that have established positions in the skin care market (Table 10.9).

The LVMH Group (Louis Vuitton and Moet Hennessey) (Paris, France) and Danial Jouvance (Carnac, France) have both invested in microalgal production sys­tems (Spolaore et al., 2006; and Kim et al., 2008). It is evident that the largest mar­ket for micro — and macroalgal cosmetics is in France, with a demand estimated at 5,000 tonnes (Kim et al., 2008). This demand will continue to escalate, with the cosmetic industry evincing more interest as research and extensive studies progres­sively highlight the benefits of microalgal extracts on skin health.

TABLE 10.9 Cosmetic Companies Producing Commercial Products Formulated with Microalgal Extracts Commercially Exploited Company Microalgae Product Effects and Benefits Exsymol SAM, Monaco Arthrospira Protulines® Anti-aging, www. exsymol. com (Spirulina) (protein-rich extract) skin-tightening effect Prevents stria formation Codif Recherche et Chlorella Dermochlorella® Stimulates collagen Nature, St Malo, France http://www. codif- recherche-et-nature. com vulgaris (extract) synthesis Supports tissue regeneration Wrinkle reduction Pentapharm, Nannochloropsis Pepha®-Tight Long — and short-term Basel Switzerland www. pentapharm. com oculata Dunaliella salina (ingredient) Pepha®-Ctive (ingredient) skin-tightening effects Stimulate cell proliferation and turnover Positively influence skin cell metabolism AGI Dermatics Inc. Anacystis Remergent™ Repairs UV damaged skin www. remergentskin. com nidulans DNA Repair Formula Resists future photo­damage through natural recuperation Company details withheld D. salina Blue Retinol™ Marestil® (Extract) Stimulates cell growth and proliferation Strong moisturizing, elasticizing, and toning complex Source: Adapted from Spolaore et al. (2006) and Kim et al. (2008).

10.2.4.2 Stable Isotope Biochemicals

Microalgae are also well suited to produce isotopically labeled compounds due to their ability to incorporate stable isotopes from inexpensive inorganic molecules into high-value isotopic organic chemicals. The ability to cultivate phototropic algae under strictly controlled conditions enables the easy incorporation of stable isotopes from inorganic carbon, hydrogen, and nitrogen sources (Pulz and Gross, 2004; Spolaore et al., 2006; Milledge, 2011). These stable isotopic compounds are used to facilitate the structural determination (at atomic level) of proteins, carbohydrates, and nucleic acids. In addition to metabolic studies (Spolaore et al., 2006), they can also be employed for clinical purposes such as gastrointestinal or breath diagnosis tests (Radmer, 1996; Pulz and Gross, 2004). Table 10.10 indicates some of the isoto­pically labeled microalgal products.

The market value of these compounds is estimated at US$13 million per year. A major distributor of such isotopic compounds is Spectra Stable Isotopes (Andover, MA; acquired by Cambridge Isotope Laboratories [CIL] in 2008) (Spolaore et al., 2006).

Innovative ways to optimize maximum microalgal biomass production and techno­logical advances for transesterification would be necessary to make microalgae more cost effective for biodiesel production and to sustain an economically viable micro­algal biotechnological industry (Figure 13.1). Improvements at various intermedi­ary stages of culturing, selection of strains of algae, harvesting, and extraction of bio-fuel production and co-products could bring down the production costs. Norsker et al. (2011) state that by optimizing irradiation conditions, mixing, photosynthetic

FIGURE 13.1 Schematic model for microalgal biotechnology.

efficiency, growth media, and CO2 costs, the overall cost of production could be reduced to Euro 0.68 per kilogram, which would be economically acceptable for using algae as feedstock for biodiesel and chemicals. Alternative metabolic pathways such as heterotrophy and mixotrophy should be explored to maximize algal growth without a shift to energetically inefficient metabolism. Service (2008) states that algae grown in dark stainless steel fermenters convert sugars to oils more efficiently. Heterotrophic and mixotrophic cultivation of microalgae in fermentation systems for commercial viability should be explored (Gladue and Maxey, 1994; Xu et al., 2006; Chi et al., 2007).

Microalgae hold great potential as a source of cheap and environmentally friendly biofuel. The total annual production of microalgae in 2004 was 5,000 tons, with global sales worth about US$1.25 billion (Pulz and Gross, 2004). However, we believe that comprehensive evaluation of select species from an integrated perspective would be of greatest benefit to commercial operations. Although Serrano’s quote (Serrano, 2010) that, “We are still like the Wright Brothers, putting pieces of wood and paper together” is in a different context, it is apt here. The rigor of microalgal biofuel research, coupled with its interdisciplinary nature, suggests that a comprehensive modeling strategy, one that accounts for numerous culture and harvest parameters and optimizes industrial processes from a perspective of cost, would be of great value. Simulation models that incorporate elements of nutrient systems, ideal culture conditions, and harvest of multiple products such as fuels and high-value nutraceu — ticals and/or recombinant proteins would be instrumental in the development of a viable bio-economy. Brown (2009) pointed out that as mass cultivation of algae for biofuels per se may not sustain microalgal technology, attention should be paid to non-fuel products and co-products as well. These co-products include carotenoids, phycolbiliproteins, astaxanthin, and eicosapentaenoic acid; additionally, algal bio­mass waste could be used as fertilizer (Donovan and Stowe, 2009).

Various processes are involved in this modeling activity. As Malcata (2010) observed, modeling exercises, instead of empirical approaches, should have biologi­cal meaning for which specific experimental data should be obtained on the optimum versus enhanced growth, metabolic cycles, assimilation efficiencies, that is, con­version of substrate into reserves, accumulation, and product sysnthesis/excretion. Scott et al. (2010) commented that there is an inadequacy of established background knowledge in this area, and there is a need to integrate biology and engineering.

The central theme rests on the predictive aspects of modeling that enable one to determine the exact quantities of the envisaged end product together with co­products. To estimate the actual quantities, we require appropriate input data regard­ing culture conditions, harvest efficiencies, and yield of co-products, as outlined above. The effective price for the microalgae-derived biofuels can be calculated by optimizing the cost functional involving several variables under appropriately for­mulated constraints. Results obtained from all stages of the process constitute the vital parameters in the mathematical model. As the process is dynamic in charac­ter, time delays do occur in a natural way, and these delays account for the process lead-time. We need to estimate these time delays, maintaining the stability of the corresponding delay-free systems. Division rates of the reaction mechanisms play a vital role in the process of restoring and/or maintaining the stability of the pro­cesses. Simulations based on realistic data will grossly help in the validation of these models. Thoroughly validated models are utilized for predicting the optimal cost of biofuel under conditions where lipid yields are maximum.

Williams and Laurens (2010) argued that a fundamental change in the approaches to production is needed, and that “biofuel-only” options may not be economically viable. They showed that 30% to 50% of primary production is lost in the produc­tion of protein and lipid, and that if lipid production is increased, then production of other valuable co-products is reduced. These authors argue that the availabil­ity of nutrients such as phosphorus and nitrogen, delivery of CO2, and the energy costs associated with sterilization and recycling of spent culture water and removal of biological contaminants, pathogens, and predators would escalate production of microalgal biomass and could be “show-stoppers.”

In conclusion, microalgal biotechnology has made rapid advances in the mass cultivation of algae and their application toward biofeed, biopharmacy, biofuel, bio­remediation, bioactive compounds, and space research. However, fewer than fifty species are utilized, while thousands remain unexplored. The potential roles of microalgae in genetic engineering and nanotechnology have increased the prospects for the next generation of “designer microalgae.” To establish algal biotechnology as an economically viable enterprise, concerted research is needed to (1) develop inex­pensive media through enrichment of wastewater; (2) isolate and culture new strains of high-yielding microalgae, preferably a consortium of extremophiles; (3) improve production systems; (4) enhance biochemical and metabolic pathways through genetic engineering; and (5) improve harvesting techniques.

Additionally, attention should be given to high-value natural and recombinant products that can be extracted from algae to enhance the profitability of biofuel oper­ations. Simulation models will serve as the foundation for industrial processes that optimize wastewater treatment systems, nutrient levels, and strategies for harvest and extraction of bioactive compounds. A robust bio-economy built on a platform of innovative microalgal technologies requires a cross-disciplinary approach among biologists, biotechnologists, molecular biologists, biochemists, engineers, chemists, bioreactor manufacturers, aquaculturists, and modelers.

ACKNOWLEDGMENTS

We are grateful to Professor Faizal Bux and Dr. Taurai Mutanda, Institute for Water and Wastewater Technology, Durban University of Technology, South Africa, for inviting us to contribute this chapter. We are most grateful to Professor John Beardall, Monash University, Clayton, Victoria, Australia, for constructive review of the manuscript. We thank Bala T. Durvasula and Dr. Ivy Hurwitz for their help with formatting the manuscript.

The research of V. Sree Hari Rao is supported by the Foundation for Scientific Research and Technological Innovation (FSRTI), a Constituent Division of the Sri Vadrevu Seshagiri Rao Memorial Charitable Trust, Hyderabad, India.

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FIGURE 2.1 Morphological diversity of microalgae: (a) Mastogloia paradoxa Grun., (b) Rhabdonema adriatium Ktz., (c) Synedra gruvei Grun.

FIGURE 2.2 Morphological diversity of microalgae: (a) Chaetoceros orientalis Schiller, (b) Nitzschia draveillensis Coste & Ricard, (c) Pleurosigma formosum Wm. Smith, (d) Coscinodiscus janischii var. arafurensis Grun., (e) Cocconeis scutellum Ehrenb., (f) Podocystis spathulata (Shadbolt) Van Heurck.

FIGURE 2.3 Morphological diversity of microalgae: (a) Actinocylus octonarius Ehrenb., (b) Biddulphia biddulphiana (Smith) Boyer, (c) Thalassionema nitzshioides Grun., (d) Rhizosolenia setigera Btw., (e) Thalassiothrix longissima Cleve & Grun.

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FIGURE 2.4 Morphological diversity of microalgae: (a) Ceratium hirundinella (Muller) Dujardin, (b) Ceratium longipeps (Bailey) Grun., (c) Ceratium trichoceros (Ehrenberg) Kofoid, (d) Gymnodinium sanguineum Hirasaka.

■ P fertilizer

■ Electrical power

■ Perspex tubing

■ N fertilizer

■ P fertilizer

■ Electrical power

■ Concrete walls

■ Steel paddle wheel

■ PVC lining

■ Water supply & treatment

FIGURE 9.2 The relative contribution of fossil energy (left) and GWP (right) to the total requirements for microalgal biodiesel production using a tubular airlift reactor (upper) and a raceway (lower). From the LCA of C. vulgaris conducted by Stephenson et al. (2010) under standard conditions. The total fossil energy requirements of 230 and 29 GJ and GWP of 13,550 and 1,900 kg CO2 per tonne biodiesel formed were estimated for the tubular reactor and raceway, respectively.

Applications and Market Segments

• Human Nutrition

Functional Foods Nutraceuticals

• Cosmeceuticals

• Animal Feed

• Pigments

о Food and Beverage Industry

о Pharmaceuticals Aquaculture Cosmetics

• Clinical and diagnostic research reagents

• Bioremediation

FIGURE 10.1 Applications of algal biomass.

CLEAN TECHNOLOGY

The most commonly used design for commercial microalgal production is the raceway pond. A raceway is an oval-shaped, single — or multiple-loop recirculation channel (Figure 5.1), usually 15 to 20 cm deep, with mixing provided through circu­lation by a rotating paddlewheel (Pulz, 2001; Brennan and Owende, 2010). Baffles are often placed in the bends of the flow channel to guide the water and facilitate mixing (Chisti, 2007). They are commonly built from concrete or packed earth,

and covered with a plastic lining. The largest raceway pond in operation is 5,000 m2, located at Earthrise Nutritionals, a commercial Spirulina producer in Southern California (Spolaore et al., 2006).

By convention, in continuous production, nutrients are introduced in front of the paddlewheel and harvesting takes place behind the wheel (Brennan and Owende, 2010). CO2 is provided by gas exchange via natural contact with the surrounding air (Singh et al., 2011). Occasionally, submerged aerators are installed to enhance CO2 absorption. Light provision is by natural sunlight. Ponds can be placed inside covered tunnels to aid in temperature regulation.

Raceway ponds incur relatively low capital investment as well as operational costs. Weekly monitoring is usually sufficient, and the main costs are in the media compo­nents and the energy consumed for mixing (Singh et al., 2011). Biomass concentra­tions are normally in the region of 0.5 g L-1, with a biomass productivity of 10 to 25 g m-2d-1 (Sheehan et al., 1998; Lee, 2001). Raceway ponds have been in use since the first commercial microalgal ventures were established, and extensive experience in their design and operation exists. Examples of the productivities obtained with various microalgal species in raceway ponds are given in Table 5.2.

The only open system to achieve very high cell densities sustainably is the cascade system developed in the Czech Republic and used for cultivation of Chlorella (Setlik et al., 1970). With a culture depth of less than 1 cm, cell densities of up to

TABLE 5.2

Examples of Productivities Achieved in Open Ponds with Various Microalgal Species

Highest Productivity

10 g L-1 Chlorella were achieved. However, the biomass productivity was compa­rable to that of raceways (25 g m-2d-1) (Lee, 2001). The system had a sloping base made of glass, which rendered it very expensive, but the use of cheaper materials could make it price competitive with raceway ponds. A similar system has been used in Western Australia, consisting of a 0.5-ha sloping, plastic-lined pond for the production of Chlorella, achieving similar biomass productivity (Borowitzka, 1999).

CO2 provision to raceway ponds through additional sparging of compressed CO2 contributed some 40% of the energy consumption and 30% GHG emissions (Clarens et al., 2010). Alternatively, co-location with industries or facilities with high CO2 emissions (such as power stations, fermentation plants, or anaerobic digester systems) may facilitate reduced contributions for effective CO2 provision. This has been demonstrated; however, it is noted that the CO2 source may influence the productivity achieved and interacts with the supply of other nutrients. Stephenson et al. (2010) modeled the impact of CO2 concentration in the gas to be compressed for sparging into either the raceway or tubular reactor system. Owing to the influence of CO2 concentration on both concentration driving force (and hence transfer rate) and on the volume of gas to be compressed, its impact is significant, with the fos­sil energy requirement nearly doubling on decreasing the CO2 concentration from 12.5% (typical of flue gases) to 9% and increasing fourfold on decrease to 5% by volume in the raceway system. The design of a low-depth carbonation sump also favors reduced energy consumption.

It is increasingly recognized that the provision of nutrients, especially combined nitrogen, to bioprocesses affects their life-cycle impact (Harding 2009; Harding et al., 2012). Lardon et al. (2009) illustrated that the provision of fertil­izer accounted for 15% to 25% of the energy requirements per unit biodiesel. This was substantially reduced under nitrogen-limited conditions (6% to 9%). Clarens et al. (2010) illustrated that 50% of the energy requirement and GWP for biomass production is attributable to nutrient provision in their raceway system. The poten­tial exists to replace these with wastewaters such as effluent from the conventional activated sludge process or source-separate urine. The former has the potential to provide a water supply simultaneously. Similar benefits can be achieved by maximizing the nutrient recycle (Stephenson et al., 2010; Richardson, 2011) and minimizing the nitrogen input required, either by optimizing the nitrogen limitation or selecting an algal species of low nitrogen content, for example, Phaeodactylum tricornutm at 0.8% N over algal species with a typical nitrogen content of 6% by mass (Richardson et al., 2012b).

Ismail Rawat, Ramanathan Ranjith Kumar, and Faizal Bux

Institute for Water and Wastewater Technology Durban University of Technology Durban, South Africa

CONTENTS

12.1 Introduction…………………………………………………………………………………………….. 179

12.2 Wastewater Characteristics…………………………………………………………………….. 181

12.2.1 Physical Characteristics………………………………………………………………. 182

12.2.2 Chemical Characteristics……………………………………………………………. 184

12.2.3 Biological Characteristics…………………………………………………………… 184

12.3 Phycoremediation…………………………………………………………………………………… 185

12.4 Algae Species Used for Phycoremediation……………………………………………… 186

12.5 High-Rate Algal Ponds (HRAPs)…………………………………………………………….. 187

12.5.1 Nutrient Removal……………………………………………………………………….. 188

12.5.2 Factors Affecting High-Rate Algae Ponds………………………………….. 190

12.5.3 Efficiency of Wastewater Treatment and Algal Growth……………. 191

12.6 Wastewater as Feedstock for Biomass Production…………………………………. 192

12.7 Economics and Energy Balance of Phycoremediation Using HRAPs……. 194

12.8 Conclusion………………………………………………………………………………………………. 195

Acknowledgments……………………………………………………………………………………………… 195

References………………………………………………………………………………………………………… 196

12.1 INTRODUCTION

The disposal of liquid and solid waste in rivers, streams, lakes, and oceans has been occurring for extended periods of time. Increasing industrialization to serve rapidly expanding urban population needs generates large amounts of wastewater that require treatment before release in order to prevent further environmental deterioration. Point source wastewater contamination has the capacity to “overload” receiving water bodies and is the most widespread threat to environmental water quality. Wastewater gener­ally contains high concentrations of organic and inorganic nutrients, which are among the main causes of irreversible ecological degradation. This disrupts the bio-system and natural recycling processes such as photosynthesis, respiration, nitrogen fixation, evaporation, and precipitation. Effective wastewater treatment and the use of reclaimed wastewater have great potential to help meet fresh water requirements for various domestic and industrial uses, thus somewhat alleviating the need for water in growing urban centers. In industrial and municipal wastewater, reduction of various chemical stacks at sources is not an easy process and is very expensive to treat by conventional treatment methods due to the demand for skilled operators, high capital investment, high operational costs, reliability etc. Complex operation of conventional treatment methods for removing chemicals does not guarantee sludge reduction. Sludge removal is one of the main challenges in sustainable wastewater treatment, but can be accomplished by the Best Available Technique (BAT) to treat the socio-economic aspect of efficient wastewater treatment. This, coupled with potential energy resource recovery, is manda­tory and necessary in exploring the feasibility of biological treatment. There has been growing worldwide interest due to decreasing water resources and increasing demand for preservation and the sustainable management of water resources (Garca et al., 2000).

Microalgal cultivation is an attractive biotechnological wastewater treatment method that has potential as an alternative method to conventional treatment. Microalgae are popular bio-resources, as appropriate microalgal technology can add a number of ben­efits to the treatment process because they have a greater capacity for the treatment of a number of wastewater contaminants. Chinnasamy et al. (2010) observed that a con­sortium of fifteen native microalgae efficiently reduced more than 96% of carpet mill treated wastewater nutrients within 72 h. Wang et al. (2010) reported rapid decreases in nitrate, phosphate, and metal levels in wastewater treatment over a short period of microalgal cultivation. Microalgal wastewater treatment is an economically viable method of wastewater treatment that has an extensive research history spanning more than 50 years (Oswald et al., 1953; Oswald, 1991; Ruiz et al., 2011). Microalgae-based wastewater removal of nutrients and/or chemicals is achieved by accumulation in, or conversion to, biomass, making it a better biotechnological method for the preservation of freshwater ecosystems (Hoffmann, 1998; Ruiz et al., 2011). Considering that inexpen­sive effluent can be used as feed for desired microalgal species to produce algae-derived products, while simultaneously removing nutrients, makes it an attractive biological system. Thus, phycoremediation technology is a promising field for applied studies such as in wastewater treatment, and biomass and biofuels production for sustainable energy.

The cultivation of microalgae for wastewater treatment is a high-quality, eco­friendly process with no secondary pollution. Reclaimed effluent produces high-value microalgal metabolites such as lipids, carbohydrates, and proteins. Microalgae are often applied in the tertiary treatment of domestic wastewater in maturation ponds, or in small — to medium-scale municipal wastewater treatment systems (Hanumantha Rao et al., 2011; Rawat et al., 2011). Technologies such as the advanced integrated wastewater pond systems (AIWPS) are commercially available (Oswald, 1991). The most common designs include facultative ponds, which are relatively deep and support surface growth of microalgae. High-rate algal ponds (HRAPs) are a hallmark technology to treat a number of wastewater streams, especially under tropical and subtropical conditions due to the availability of sunlight utilized by microalgae for photosynthesis (Phang et al., 2000; Mustafa et al., 2011). Shallow ponds depend on mechanical mixing for maxi­mum algae production and removal of biological oxygen demand. HRAPs are the most
cost-effective reactors for liquid waste management and capture of solar energy, and the capture of atmospheric carbon dioxide, and are used in the treatment of animal wastes (Narkthon, 1996). HRAP wastewater treatment can be highly efficient in reducing bac­teria, biological oxygen demand (BOD), and nutrient levels by integrated approaches to recycling wastes. Phycoremediation can be used in the process as a second step after initial anaerobic treatment of high organic wastewater to yield a significant reduction in influent organic matter, such as nitrogen and phosphorus. Harvested microalgae are rich in nutrients such as nitrogen, potassium, and phosphorus, which can be used for animal feed, etc. (Ogbonna et al., 2000; Olguin, 2003; Rawat et al., 2011). Therefore, HRAPs are very appropriate for sanitation in small rural communities because of their simplicity of operation in comparison to conventional technologies such as the acti­vated sludge process. This chapter critically evaluates phycoremediation HRAPs for removal of high organic strength nutrients by means of enriched microalgae.

Floatation is a separation process based on the attachment of air bubbles to solid particles. The resulting flocs float to the liquid surface and are harvested by skim­ming and filtration. The success of flotation depends on the nature of suspended particles (microalgal cells in harvesting process). Air bubbles drift up the smaller particles (<500 pm) more easily (Matis et al., 1993). Also, the lower instability of suspended particles results in relatively higher air-particle contact. The attachment of air bubbles also depends on the air, solid, and aqueous phase contact angle. The larger the contact angle, the greater the tendency of air to adhere (Shelef et al., 1984). Dissolved air flotation (DAF), electrolytic flotation, and dispersed air flotation are the commonly used flotation techniques according to the method of bubble pro­duction. Dissolved air flotation is the most widely used method for the treatment of industrial effluent. Van Vuuren et al. (1965) performed a study on flotation and reported that flocculation requires several hours of sedimentation, while flotation shortens the duration to only a few minutes. The DAF procedure by chemical floc­culation is reported to recover up to 6% (w/v) algal biomass slurries from algae cul­ture (Bare et al., 1975). Although flotation has been used by several researchers as a potential harvesting method, there is only limited evidence of its technical and economic viability.

Carotenoids are colored, lipid-soluble compounds that occur in higher plants, micro­algae, as well as in nonphotosynthetic organisms (Del Campo, 2007; Takaichi, 2011). Carotenoids contribute to light harvesting, maintenance of structure, and functioning

Applications and Market Segments

• Human Nutrition Functional Foods Nutraceuticals

• Cosmeceuticals

• Animal Feed

• Pigments

о Food and Beverage Industry

о Pharmaceuticals Aquaculture Cosmetics

• Clinical and diagnostic research reagents

• Bioremediation FIGURE 10.1 (See color insert.) Applications of algal biomass.

TABLE 10.1 Global Production of Algal Biomass for Commercially Relevant Algal Genera Production Genus (tonnes y-1 dry weight) Country Applications and Products Spirulina 3,000 China, India, U. S., Human and animal nutrition, Myanmar, Japan, SA phycobiliproteins, cosmetics Chlorella 2,000 Taiwan, Germany, Human nutrition, aquaculture, Japan cosmetics Dunaliella 1,200 Australia, Israel, Human nutrition, cosmetics, U. S., China, SA p-carotene Nostoc 600 China Human nutrition Aphanizomenon 500 U. S. Human nutrition Haematococcus 300 U. S., India, Israel Aquaculture, astaxanthin Source: Adapted from Pulz and Gross (2004), Spolaore et al. (2006); Milledge (2011).

of photosynthetic complexes in plants and microalgae (Pulz and Gross, 2004; Del Campo, 2007). They occur widely in nature and are responsible for many of the brilliant red, orange, and yellow colors of edible vegetables and fruits and some aquaculture animals.

Microalgae combine properties of higher plants with some properties of prokary­otes. This combination represents the rationale for using microalgae for the pro­duction of carotenoids and other products (Del Campo, 2007; Guedes et al., 2011) instead of using plants or prokaryotes. Furthermore, the production of carotenoids by microalgae can be easily maximized by manipulating growth conditions. Under unfavorable growth conditions, microalgae produce high amounts of carotenoids, such as P-carotene, astaxanthin, and canthaxanthin (Orosa et al., 2000).

Microalgal wastewater treatment using microalgae with the production of biomass as a by-product is not a new concept. However, it occurs only on a minor scale in waste stabilization ponds and HRAPs. Wastewater treatment using HRAPs has the potential to produce large amounts of biomass that can be used for a variety of applications, including the production of renewable fuels, fertilizer, animal feed, etc. (Rawat et al., 2011). Recent studies have suggested that the use of wastewater as a substrate for biofuel production may make the process economically viable (Brennan and Owende, 2010; Boelee et al., 2011; Cho et al., 2011). Focusing the growth of microalgae on biomass productivity rather than lipid productivity may be beneficial as larger amounts of biomass improve the viability of conversion to alternate fuels (Pittman et al., 2011). Microalgal biomass to biofuels conversion may be carried out by several methods depending on the biomass characteristics (e. g., lipid or carbohy­drate content) (Garcia et al., 2006; Rawat et al., 2011). The yields of biomass from HRAPs depend on the type of effluent being treated with specific regard to nutrient content. Table 12.2 summarizes growth and lipid productivity of microalgal species on a variety of wastewater types. Piggery waste effluent treatment by HRAPs has potential productivities of up to 50 t ha-1yr-1 (Rawat et al., 2011).

Maximum algal productivities in HRAPs can be achieved by countering rate — limiting and inhibitory conditions. Carbon is often a rate-limiting substrate and may be alleviated by the addition of CO2. This addition serves a dual role in the provi­sion of carbon and a method of pH control. The addition of CO2 has been shown to double algal productivity at the laboratory scale and increase productivity by 30% in a pilot-scale HRAP (Park et al., 2011a). Biomass grown at the Lawrence wastewater treatment plant showed algal productivities ranging from 5 to 16 g m-2d-1 and average lipid contents of 10% without the addition of CO2. With the addition of CO2, produc­tivities were expected to be 25 g m-2d-1 (Sturm and Lamer, 2011). However, it must be considered that addition of excess CO2 leads to a decrease in pH. A pH maintained at a maximum of 8 inhibits physico-chemical processes of nutrient removal such as volatilization of ammonia and phosphate precipitation (Craggs, 2005). But this is not necessarily a negative point, as the increase in assimilation by biomass production offsets the losses on physico-chemical removal. Furthermore, it enables the recycling of nutrients that would have been otherwise lost. Ammonia volatilization accounts for approximately 24% nitrogen loss in HRAPs without pH control (Park et al., 2011a).

TABLE 12.2

Biomass and Lipid Productivities of Microalgae Grown on Various Wastewater Streams

		Biomass (DW) Productivity	Lipid Content	Lipid Productivity
Wastewater Type	Microalgal Species	(mg L-1d-1)	(%DW)	(mg L-1d-1)
Municipal (primary treated)	nd	25a	nd	nd
Municipal (centrate)	Chlamydomonas reinhardtii (biocoil grown)	2000	25.25	505
Municipal (secondary treated)	Scenedesmus obliquus	26b	31.4i	8і
Municipal (secondary treated)	Botryococcus braunii	345.6c	17.85	62
Municipal (primary treated + CO2)	Mix of Chlorella sp., Micractinium sp., Actinastrum sp.	270.7d	9	24.4
Agricultural (piggery manure with high NO3-N)	Botryococcus braunii	700e	nd	69
Agricultural (dairy manure with polystyrene foam support)	Chlorella sp.	2.6 g m-2d-1	9і	230і mg m-2d-1
Agricultural (fermented swine urine)	Scenedesmus sp.	6f	0.9і	0.54i
Agricultural (anaerobically digested diary manure)	Mix of Microspora willeana, Ulothrix zonata, Ulothrix aequalis, Rhizoclonium hieroglyphicum, Oedogonium sp.	5.5 g m-2d-1	nd	nd
Agricultural (swine effluent, maximum manure loading rate)	R. hieroglyphicum	10.7 g m-2d-1	0.7і	72і mg m-2d-1
Agricultural (swine effluent, +CO2, maximum manure loading rate)	R. hieroglyphicum	17.9	1.2і	210 mg m-2d-1
Agricultural (digested dairy manure, 20x dilution	Chlorella sp.	81.4®	13.6i	11і

TABLE 12.2 (Continued) Biomass and Lipid Productivities of Microalgae Grown on Various Wastewater Streams Biomass (DW) Lipid Lipid Productivity Content Productivity Wastewater Type Microalgal Species (mg L-1d-1) (%DW) (mg L-1d-1) Agricultural Mix of Chlorella sp., 59h 29 17 (dairy wastewater, Micractinium sp., 25% dilution) Actinastrum sp. Industrial (carpet mill, Botryococcus braunii 34 13.2 4.5 untreated) Industrial (carpet mill, Chorella saccharophila 23 18.1 4.2 untreated) Industrial (carpet mill, Dunaliella tertiolecta 28 15.2 4.3 untreated) Industrial (carpet mill, Pleurochrysis carterae 33 12 4 untreated) Artificial wastewater Scenedesmus sp. 126.54 12.8 16.2 Source: From Rawat et al. (2011). Note: nd — Not determined. a Estimated from biomass value of -1000 mg L-1 after 40 days. b Estimated from biomass value of 1.1 mg L-1 h-1. c Estimated from biomass value of 14.4 mg L-1 h-1. d Estimated from biomass value of 812 mg L-1 after 3 days. e Estimated from biomass value of 7 g L-1 after 10 days. f Estimated from biomass value of 197 mg L-1 after 31 days. g Estimated from biomass value of 1.71 g L-1 after 21 days. h Estimated from lipid productivity and lipid content value. i Fatty acid content and productivity determined rather than total lipid.

The counting chamber methods are well established and frequently applied for micro­algal enumeration due to their low cost and easy application. The three common types of counting chamber methods for microalgae enumeration are the (1) Sedgewick — Rafter counting slide, (2) Palmer-Maloney counting slide, and (3) haemocytometer counting slide (LeGresley and McDermott, 2010). The three methods require sam­ples with high cell densities. The presence of contaminating particles in the same size range as the algae and failure of cells to separate after cell division may be possible sources of erroneous counts (Coutteau, 1996). Table 4.1 compares the merits and drawbacks as well as fundamentals of the three counting chamber methods.