Nucleus A major mechanism of nuclear gene regulation is transcriptional control. Strategies to enhance heterologous gene expression, which occurs at relatively low levels compared to expression in the chloroplast, are the search for more effective promoters (Rasala et al. 2013) and the enhancement of translation efficiency through codon optimisation (Fuhrmann et al. 1999, 2004; Heitzer and Zschoernig 2007; Mayfield and Kindle 1990). The glycosylation patterns of nuclear expressed and secreted proteins from C. reinhardtii remain to be resolved. Studies on the post­translational machinery of this alga would be helpful to exploit this as an option of algal protein production.

Chloroplast Ensuring high heterogeneous gene expression requires the identifica­tion of endogenous transcriptional and translational regulatory elements (Harris et al. 2009; Marin-Navarro et al. 2007; Purton 2007). Although chloroplast gene expression is usually regulated at the translational level (Barnes et al. 2005; Eb — erhard et al. 2002; Nickelsen 2003; Rasala et al. 2010, 2011; Zerges 2000), choice of promoter and 5′ UTRs sequences is of importance due to potential feedback regulations which can interfere with the heterologous protein expression and mRNA stability (Gimpel and Mayfield 2013; Hauser et al. 1996; Manuell et al.

2007) . The 5′ UTRs are believed to regulate ribosome association, transcript sta­bility and the rate of translation (Barnes et al. 2005; Marin-Navarro et al. 2007; Salvador et al. 1993; Zou et al. 2003) while 3′ UTRs influence mRNA stability (Herrin and Nickelsen 2004; Lee et al. 1996; Monde et al. 2000; Stern et al. 1991) or may interact with 5′ UTRs (Katz and Danon 2002).

Mitochondria In photosynthetic organisms, mitochondria are the organelle with the greatest diversity in size and structure, ranging from 15 kb linear DNA mole­cules in Chlamydomonas to 1.0 Mb in angiosperms. To date, Chlamydomonas is the only photosynthetically active organisms for which mitochondrial DNA has been altered, which significantly limits insights into translational and transcriptional control. Attempts have been made with plant mitochondria to utilise in vitro DNA and RNA import, and electroporation of isolated mitochondria has been used to gain further information about transcription and post-transcriptional processing (Remacle et al. 2012).

Flocculation is based on the addition of materials (flocculants) or changing the medium in such a way the cells are attracted to each other and therefore more rapidly settle to the bottom of a holding tank. This is really the result of coagulation and then flocculation. Coagulants destabilize the charges and surface properties of the cells in suspension, so they do not resist agglomeration. Flocculants focus on stimulating the formation of larger aggregates from the destabilized algal cells.

Autoflocculation, chemical flocculation, bioflocculation, and electroflocculation are all examples of processes that rely on flocculation to aggregate and speed the removal of algal cells. At scale, all methods for flocculation require large amounts of space and are expensive due to costs of coagulants and/or flocculants and operators (Bosma et al. 2003) or other chemical additions (e. g., pH adjustment in autoflocculation).

Nutrients in the form of commercial fertilisers are an expensive input for mass production of microalgae biomass (Collet et al. 2010), particularly nitrogen and phosphorous (Fenton and Ohuallachain 2012; Lyovo et al. 2010; Vaccari 2009). Large nutrient requirement can increase cost of microalgae biofuel production and compete with agricultural demand (Erkelens et al. 2014; Fenton and Ohuallachain 2012). Fertiliser prices are highly dependent on fossil fuels price’s (Fenton and Ohuallachain 2012; Vaccari 2009). Increase in fossil fuel price coupled with the higher worldwide agricultural demand resulted in increased fertiliser costs (Fenton and Ohuallachain 2012; Stephans et al. 2010; Vaccari 2009; Ward et al. 2014). Therefore, nutrient recovery from residual and waste products from the microalgae production is essential to allow sustainable product development (Erkelens et al. 2014; Sialve et al. 2009; Stephans et al. 2010). Anaerobic digestion can offer a solution to the fertilisers input problem by recycling extracted biomass and any by­products (Erkelens et al. 2014). Anaerobic digestion of algal biomass produces a clear liquid digestate that is nutrient rich containing both nitrogen and phosphorous (Stephans et al. 2010). Anaerobic digestion digestate nutrient content of 546­2940 mg/L ammonia nitrogen and 141-390 mg/L phosphorous have been reported from anaerobically digested microalgae (Collet et al. 2010; Erkelens et al. 2014; Ward et al. 2014; Zamalloa 2012). A further benefit of the integration of anaerobic digestion into microalgae production is the ability to utilise the microalgae cultures to purify the biogas produced from anaerobic digestion (Converti et al. 2009; Green et al. 1995a). The concentration of methane in biogas produced from microalgae is in the range of 30-50 % (Sialve et al. 2009); generally too low to utilise in its current form, and purification of the biogas is needed before utilisation (Vergara-Fernandez et al. 2008). Due to the low solubility of methane and high solubility of CO2, the uptake of CO2 in microalgae cultures is able to purify the biogas to a higher energy density, with the added benefit of stripping other gasses such as sulphur and ammonia (Green et al. 1995a; Ward et al. 2014). As methane has been shown to be non­detrimental to microalgae growth, the dual purification of biogas as a consequence of supplying microalgae cultures with additional nutrient in the form of CO2 achieves multiple productivity benefits (Green et al. 1995a; Sialve et al. 2009).

Several unicellular green algae are capable of generating hydrogen through their [FeFe]-hydrogenase enzyme by reducing water protons to molecular hydrogen. However, given the sensitivity of [FeFe]-hydrogenase to oxygen, which is gener­ated by photosystem II (PSII), new approaches have been developed for increasing the practical application of microalgae for biohydrogen production (Laurinavichene et al. 2008). For instance, higher hydrogen production efficiencies were achieved by growing the microalgal cells under sulfur-deprived conditions (Melis et al. 2000). Sulfur deprivation causes partial inactivation of PSII, which is responsible for O2 generation, resulting an enhanced synthesis of [FeFe]-hydrogenase enzyme (Laurinavichene et al. 2008).

Immobilization processes have been proposed by several researchers for enhancing hydrogen production by sulfur-deprived microalgae and also allowing an easy exchange step between “sulfur-replete” and “sulfur-depleted” stages of the experiment (Laurinavichene et al. 2006, 2008). Several challenges require addressing for scaling up the current hydrogen production systems, while immo­bilization processes offer an alternative approach to the current technology. Immobilized cells were also reported to have higher light utilization efficiencies per area and higher cell densities (Kosourov and Seibert 2009).

In a study by Kosourov and Seibert (2009), C. reinhardtii cells were immobi­lized inside alginate films for the photoproduction of hydrogen. The cells were previously deprived of sulfur and phosphorus nutrients before being entrapped inside the alginate films. They observed higher cell densities and specific hydrogen production rates after the immobilization process. An immobilization strategy also provided easy protection of the hydrogenase enzyme from oxygen inhibition, yielding higher hydrogen production rates compared to the free cells.

Laurinavichene et al. (2006, 2008) used immobilized C. reinhardtii cells on a fiber glass matrix under sulfur-deprived conditions and observed a prolonged hydrogen production phase for the immobilized cells, while the specific hydrogen production rate was similar to the free-cell counterparts. In another study, algal cells were immobilized on fumed silica particles, which had similar hydrogen production rates with the suspended cultures (Hahn et al. 2007). Song et al. (2011) recently used agar-immobilized Chlorella sp. cells for a two-stage cyclic hydrogen pro­duction involving the oxygenic photosynthesis followed by anaerobic incubation under sulfur-deprived conditions.

Stephen R. Lyon, Hossein Ahmadzadeh and Marcia A. Murry

Abstract This chapter develops the principles and rationale for an algae-based biofuel production coupled to bioremediation of municipal and agricultural wastewaters. A synergistic model for algal wastewater treatment is proposed, which addresses several economic bottlenecks to earlier algal systems and promotes value — added products, including a high-quality effluent in addition to biodiesel to improve the economic feasibility of algal biofuels. Finally, we review candidate species for full-scale algae production ponds based on algal structure, physiology and ecology, and methods for extraction of algal oils for biodiesel production and coproducts. The dominant strains of algae that are commonly found in wastewater ponds, including Euglenia, Scenedesmus, Selenastrum, Chlorella, and Actinastrum, are suggested as candidates for large-scale culturing based on their ability to strip nutrients and organic matter from wastewater, grow rapidly, and produce a sig­nificant level of algal oil. Oil extraction by supercritical fluid extraction (SFE) is discussed as an efficient means of isolating algal oil and other commercially important high-value compounds from algal biomass. Together with water and CO2 reclamation, such products may shift the economics of algal biomass production to allow production of low-value commodities including biodiesel and biogas.

S. R. Lyon

AlgaXperts, LLC, Milwaukee, WI, USA H. Ahmadzadeh (H)

Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran e-mail: h. ahmadzadeh@um. ac. ir

M. A. Murry

California State Polytechnic University, Pomona, CA, USA M. A. Murry

Sinai Technology Corporation, Los Angeles, MI, USA © Springer International Publishing Switzerland 2015

N. R. Moheimani et al. (eds.), Biomass and Biofuels from Microalgae,

Biofuel and Biorefinery Technologies 2, DOI 10.1007/978-3-319-16640-7_6

6.1 Big Picture

The global biofuels market has made the transition from concept to multibillion-dollar reality in the past twenty years. The first-generation biofuels included ethanol and biodiesel. Ethanol is produced from the fermentation of sugar or starch-rich crops such as sugar cane and corn and distilled to yield pure ethanol. More recently, a variety of novel cellulase and xylanase enzymes have been identified from symbiotic and free — living microbes for use in the saccharification process allowing a greater fraction of the plant biomass used for ethanol production (Gladden et al. 2011). Biodiesel is primarily derived from the extraction and transesterification of triacylglycerols (TAGs) or triglycerides from various oil-bearing plants such as canola or jatropha or from the extraction of oil from soy, corn, and oil palm. Traditionally, biodiesel is made by transesterification of extracted TAGs producing fatty acid methyl esters (FAMEs) that can be used in diesel engines without modification and glycerol as a co-product.

Global ethanol production has grown to 22 billion gallons (US) in 2011 with the USA and Brazil contributing more than 65 % of total production (Renewable Fuels Association 2012). Biodiesel production is also growing rapidly, albeit from a smaller starting point. In 2008, the global biodiesel market was worth $8.6 billion (US) and is expected to grow to $12.6 billion (US) in 2014 (Davis et al. 2013). Although these first-generation biofuels represent a rather small contribution to the transportation fuel industry, they have come under criticism for competing with food production for arable land, nutrients, and water. Early biofuel programs had a negative impact on global food supplies with regard to soy, corn, and other grains or the destruction of tropical rain forests with regard to sugar cane and oil palm. In addition, terrestrial crops require months or in the case of oil palm years of growth before they can produce a harvestable crop. In response to the difficulties associated with the first-generation biofuels, interest in algae-based biofuels has been renewed and wide-spread efforts have gone into solving some of the technical problems associated with cost-efficient large-scale algal biomass production.

Algae are defined as a group of photosynthetic organisms, ranging from uni­cellular to multicellular forms, which lack true roots, stems, and leaves character­istic of terrestrial plants. Photosynthetic microorganisms, generally referred to as microalgae, represent a complex and diverse array of life forms that vary greatly in their metabolic capabilities, environmental adaptations, and morphology. The four common characteristics that are of significance with regard to this chapter are that they are small, autotrophic (i. e., they take up carbon dioxide to produce their own carbon compounds for metabolic purposes in sunlight), some are mixotrophic (i. e., they can assimilate a variety of carbon compounds in the absence of sunlight), and they produce varying amounts of oil (lipid) in the form of diglycerides and tri­glycerides. Algal oil is similar in structure and molecular weight to the oils extracted from the terrestrial plants described above for the production of biodiesel or as a feedstock for industrial chemicals that have a higher market value.

Lipid production in microalgae is species specific and influenced by environ­mental conditions. Oil content in pure cultures of microalgae can range from 1 % to over 50 % of the dry weight. In addition, some algae can double their biomass in as little as 3.5 h in the laboratory and 24 h in outdoor ponds. Oil seed plants require an entire season for maturation of oil-rich seeds, which, in turn, comprise only a relatively small fraction of plant biomass. Algae lack non-photosynthetic structures (i. e., roots and stems), and since microalgae are unicellular and float in the water column, they have no need for the massive amounts of structural cellulose found in land plants. Furthermore, the photosynthetic efficiency of microalgae can theoret­ically reach up to 12 % (Oswald 1963; Zelitch 1971; Weissman and Goebel 1987), while terrestrial plants at mid-latitudes convert less than 0.5 % of solar energy into biomass (Li et al. 2008). Thus, productivity of microalgae per unit of land use can yield 7-20 times greater biomass than soy or corn and many strains can grow in saline or wastewaters. Taking these factors into consideration, it is easy to see why the potential difference in biomass/oil production between plant-based and algae — based biodiesel is so great. However, while it is true that some algae can accumulate biomass faster than terrestrial crops and tend to store excess carbon as lipids rather than structural carbohydrates, this frequently cited point distracts attention from the proper metric, which is total cost of oil production.

Macroalgae (i. e., seaweed) have been commercially produced for centuries. About 1.8 x 106 t of seaweed is produced commercially throughout the world. Until recently, the annual production of microalgae amounted to roughly one-hundredth the amount of commercially produced seaweed on an annual basis (Neori 2008). While many commercial microalgae production operations have been established in the last 40 years to produce high-value phytochemicals (e. g., beta-carotene, asta — xanthin, and zeaxanthin), pharmaceuticals, feed for mariculture applications, and health food supplements (see Spolaore et al. 2006, for a review), the economic feasibility of producing algae biomass for low-value commodities including bio­fuels remains uncertain. Significant improvements in several key technologies, including strain selection, best cultivation practices, maintaining selected species in ponding operations, harvesting, and oil extraction, are needed to advance the economics of algae-based biofuel production. Considerable progress has been made over the past six years to develop and commercialize missing elements in the algae biofuels production chain. Innovative algae dewatering technology (AlgaeVenture Systems, Inc., Marysville, Ohio) and wet extraction and oil conversion technologies (SRS Energy Solutions, Inc., Dexter Mich.; Genifuels, Inc., Salt Lake City, Utah) are in demonstration phase.

While continued research and development of these technologies will improve the economics of algae biofuels, major economic limitations could be overcome in the short term by integrating biofuel production with wastewater treatment, to provide additional economic and environmental benefits. In synergy with biofuel production, algae-based wastewater treatment is a lost-cost, simple process com­pared to conventional wastewater systems, and algae systems have about 50 % lower energy consumption compared to conventional mechanical treatment tech­nologies (Downing et al. 2002; Lundquist et al. 2010; Craggs et al. 2012).

Forward genetics refers to the process of identifying a phenotype and then char­acterizing the genes that are involved in the phenotype’s biological pathway or process through the screening of populations of a modified organism (Lawson and Wolfe 2011). Forward genetics was successfully used to determine genes involved in metabolic and regulatory pathways in Chlamydomonas. The availability of a Chlamydomonas linkage map and a protocol for genetic crossing, along with the complete genome sequence, has boosted the gene identification process (Gonzalez — Ballester et al. 2011). Modification of an organism for screening purposes can be achieved through random mutagenesis (UV irradiation or chemically induced) to generate mutant populations to be mapped. An alternative approach to obtain a mutant population in various microalgal species is insertional mutagenesis. Inser­tion of a DNA fragment into the coding region of a particular gene results in the disruption of a protein-coding sequence and the loss of function of that gene (Vuttipongchaikij 2012).

Reverse genetics involves starting with a known gene and then disrupting the function of that gene to produce a phenotype and gain insight into what that gene does. Besides disruption, overexpression of a gene is another option. The gene sequences within a given organism need to be known, and the goal here is to define the sequences’ functions. In reverse genetics, mutations are made in specific genes or gene products to determine their effect on the organism, and from that determine the gene’s function. The availability of complete genome sequences combined with reverse genetics can allow every gene to be mapped to a function (Ahringer 2006). Using available characterized or annotated genes, improved microalgal strains can be generated through reverse genetics by gene silencing or random mutagenesis or even through gene modification by homologous recombination (Vuttipongchaikij 2012). Gene silencing by RNAi has also been used successfully for reducing gene expression levels in microalgal systems and for controlling gene function (Rohr et al. 2004). RNAi is proving to be a very useful tool for reverse genetics. Still, the most dramatic way to reduce gene activity is to eliminate the gene entirely by deleting it from the genome. This can be achieved through homologous recombi­nation (see Sect. 3.3).

Flocculation can be induced by several mechanisms (Fig. 12.4). A first mechanism is charge neutralization, or neutralization of the negative surface charge by adsorption of oppositely charged ions such as Mg+2 or Ca+2 to the cell surface. When the surface charge of the cell is neutralized, the electrostatic repulsion between the cells disap­pears. As a result, cells can approach each other and will connect through Van der Waals interactions resulting in the formation of flocs. In charge neutralization, the flocculant dose is a linear function of the surface area that needs to be neutralized. When dosage exceeds the optimal dose for charge neutralization, the surface charge

may shift from negative to positive, and the cell suspension may become stabilized again; a phenomenon that is known as dispersion restabilization. Flocs formed by charge neutralization are often small and unstable (Gregory 2006).

A second flocculation mechanism is bridging by polymers. A polymer carrying positive charges, for example, chitosan, may bind to the negatively charged cell surface with the polymer chain extending from the cell surface. If the polymer connects to another cell, bridges can be formed between cells and flocs are formed. Longer polymers and weakly charged polymers are often more efficient than shorter and highly charged polymers. When a high polymer dose is used, the cell sus­pension may become stabilized again. This dispersion restabilization can be due to an inversion of the surface charge from negative to positive, but it can also be due to steric hindrance caused by the polymer chains that are associated with the surface (steric stabilization). Flocs formed by bridging are often large and stable (Fellows and Doherty 2006; Bolto and Gregory 2007).

A third mechanism of flocculation is the electrostatic patch mechanism. Here, positively charged polymers connect to the negatively charged cell surface and form patches on the cell surface, where the surface charge is inversed from negative to positive. Positively charged patches on one cell can connect to a negatively charged patch on another cell and flocs can be formed. Short and highly charged polymers often cause flocculation by the electrostatic patch mechanism (Bolto and Gregory 2007).

The final mechanism reviewed is sweeping flocculation or flocculation by enmeshment. In this mechanism, the flocculant forms a precipitate that enmeshes the microalgal cells. The cells then will settle or flocculate together with the precipitate. The precipitate often works as a ballast that facilitates the settling of the cells (Yahi et al. 1994). In sweeping flocculation, the flocculant dose is often independent of the concentration of particles that is flocculated. Flocs formed by a sweeping mechanism are often more stable than flocs formed by charge neutralization.

It is often not straightforward to conclude which mechanism is responsible for flocculation. A polymer flocculant may induce flocculation through charge neu­tralization, bridging or electrostatic patch neutralization. A metal salt coagulant may induce flocculation through charge neutralization or a sweeping mechanism (Duan and Gregory 2003). In many studies on flocculation of microalgae, one or a combination of mechanisms is often proposed, but few studies so far have dem­onstrated unequivocally which mechanism is responsible for inducing flocculation.

Completely or partially eliminating the harvesting step is a direct way to reduce the cost of harvesting on the process, but is only valuable if it does not impact the downstream processing of products and co-products. There are several ways that are being explored to truncate or eliminate harvesting in algal biofuel production.

Mass culture in a biofilm. Supplying a suitable surface on which to mass culture algae without suspension in a dilute culture medium would allow the direct harvest of concentrated algae rather than trying to separate a dilute suspension of algal cells from their culture medium. In theory, this method would also allow better access to nutrients and sunlight.

A biofilm-based system is currently being commercialized by BioProcessAlgae LLC. Their Grower Harvester™ system provides a substrate on which algae are inoculated and allowed to grow under suitable conditions and then removed in a more concentrated form using a stream of water to remove the cells from the substrate (http://www. bioprocessalgae. com/technology/). Little data are publically available at this time on the economics of this process.

Directly process high moisture content biomass. Elimination or partial elimi­nation of secondary harvesting would mean that the algal pastes do not have to be highly concentrated, leaving a large amount of water with the algal cells (>20 % moisture). A number of different technologies are applicable to process high moisture algal pastes, including hydrothermal liquefaction (Biddy et al. 2013), catalytic hydrothermal gasification (Biller et al. 2011), supercritical methanol conversion to biodiesel (Patil et al. 2011), and hydrothermal carbonization (Heil — mann et al. 2011). All of these processes either require the presence of water for conversion to biofuels or can tolerate high levels of moisture in the biomass without excessive parasitic energy loss. While it is beyond this review to discuss all of these methods, one example is the use of supercritical methanol conversion to biodiesel on algal paste. Open pond-grown Nannochloropsis sp. biomass at <10 % solids was directly converted to fatty acid methyl esters suitable for use in biodiesel (Patil et al. 2011).

Direct harvesting of biofuel or bioproducts. An indirect approach has also found favor in the microalgal industry where the biofuels are not contained within the algal cells but secreted into the medium so that the products are harvested without having to harvest the algal biomass itself (opening the prospect of reuse of the algal biomass for additional product generation).

One unique alga, B. braunii, accumulates lipid on the exterior of the cell as it becomes senescent or stressed. This alga produces some very long chain and unique lipids but grows very slowly. A suggestion that continuous harvest using a bio­compatible solvent in an aqueous/solvent bioreactor has recently been proposed that would allow direct harvesting on a continuous basis to lower costs and offset the slow growth of the biomass (Moheimani et al. 2014; Zhang et al. 2011).

An example of this approach is that currently applied by Algenol for the pro­duction of ethanol from cyanobacteria (bluegreen algae). Secretion of algal lipid into the medium could allow continuous culturing of the algal biomass while removing product from the culture medium through phase separation. This has been demonstrated in bacteria (Sauer and Galinski 1998) and has been proposed for diatoms and other algae (Ramachandra et al. 2009). The company Synthetic Genomics reports that they have developed algal cells that secrete oil in a contin­uous manner to produce an algal biocrude (www. syntheticgenomics. com/what/ renewablefuels. html) and have filed patents protecting the idea (Roessler WO2009076559). Other companies have similar ideas being applied to cyano­bacteria, such as Joule Unlimited with enclosed PBRs and excreted volatile organics, although they are not publishing their methods, so it is hard to calculate the economics.

Aside from mineral elements and nitrogen sources, some wastewaters contain high concentrations of organic carbons for mixotrophic/heterotrophic cultivation of microalgae. Thus, some wastewaters can be used as both carbon and nitrogen sources for cultivation of microalgae. Cultivation of microalgae in wastewater for biodiesel production is highly desirable since it leads to a significant reduction in the production costs and reduction in the demand for freshwater with the con­comitant removal of various contaminants, such as phosphorus, nitrogen, heavy metals, and pathogens from the wastewater.

In addition to carbon, nitrogen, and phosphorus, microalgae also require micronutrients for growth and oil production. Micronutrients required in trace amounts include silica, calcium, magnesium, potassium, iron, manganese, sulfur, zinc, copper, zinc, nickel, lead, chromium, and cobalt (Bao et al. 2008; Ortiz Escobar and Hue 2008; Faridullah et al. 2009; Vu et al. 2009). These nutrients are usually added through the addition of commercial fertilizers, which substantially increase production costs. The concentrations of these essential micronutrients rarely limit algal growth when wastewater is used (Knud-Hansen et al. 1998). Furthermore, many wastewaters such as poultry litter, slaughter house wastes, dairy effluents, swine wastes, municipal wastewater, and effluents from anaerobic digesters are rich in organic nutrients. In addition to supplying these nutrients, the cultivation of microalgae in wastewaters is an efficient method of wastewater treatment (Ogbonna et al. 2000). Hodaifa et al. (2008) recorded 67.4 % reduction in BOD with S. obliquus cultured in diluted (25 %) industrial wastewater from olive oil extraction. Wang et al. (2009) also reported that wastewaters from different stages of treatment are good for cultivation of Chlorella sp. with efficient removal of N, P, and COD. A consortium of 15 native algal isolates removed more than 96 % nutrients from wastewater (Chinnasamy et al. 2010). However, there are variations in the composition of wastewaters and each may be suitable for culti­vation of only a few strains of microalgae for some specific purposes. Furthermore, most wastewaters are opaque, limiting light penetration, and thus are not suitable for photoautotrophic culture.

Types of wastewaters investigated for microalgae cultures include municipal, industrial, and agricultural wastewaters (Jiang et al. 2011). For example, poultry litter contains approximately 3.3 % nitrogen and 2.6 % phosphorus and cell growth promoters, such as glycine, are released from poultry manure on decomposition (Schefferle 1965). The composition of poultry manure depends on the type of feed used. For example, according to Magid et al. (1995), some common nutrients in poultry manure include (g/Kg) potassium 37.5, phosphate 25.5, and nitrogen 55.7. Nitrogen is normally in the form of uric acid, and about 66 % can be available on decomposition (Ruiz et al. 2009).

It has further been reported that various species of microalgae were cultivated in POME and biomass productivity varied from 2.9 to 8.0 mg/L/day and the oil content ranged from 21.34 to 30.83 % (Putri et al. 2011; Nwuche et al. 2014). One problem of POME as a medium for microalgae is the high COD content, dark color of tannic acid, and high impurity. This could be solved by anaerobic digestion to significantly reduce COD, TS, TSS, T-nitrogen, and orthophosphate (Habib et al.

2003) . Rubber Mill Effluent consists of latex washings and a solution containing proteins, sugars, lipids, and inorganic and organic salts. The high level of ammonium and other plant nutrients make it a good medium for algal growth (Azimatun-Nur and Hadiyanto 2013). Fermented cocoa bean mill effluent, also called cocoa-sweating effluent, contains several sugar residues and micronutrients (Syafila et al. 2010). The final lipid content of the culture with feeding of effluent from stably operated anaerobic continuous-flow stirred-tank reactor was 27 ± 1.11 % after 168-h cultivation in flasks, which was higher than the value obtained with glucose of the same COD concentration (Wen et al. 2013).

Mohammad J. Raeesossadati, Hossein Ahmadzadeh, Mark P. McHenry and Navid R. Moheimani

Abstract Various microalgae species have shown a differential ability to biore­mediate atmospheric CO2. This chapter reports biomass concentration, biomass productivity, and CO2 fixation rates of several microalgae and cyanobacteria species under different CO2 concentrations and culture conditions. Research indi­cates that microalgal species of Scenedesmuss obliquss, Duniella tertiolecta, Chlorella vulgaris, Phormidium sp., Amicroscopica negeli, and Chlorococcum littorale are able to bioremediate CO2 more effectively than other species. Fur­thermore, coccolithophorid microalgae such as Chrysotila carterae were also found to effectively bioremediate CO2 into organic biomass and generate inorganic CaCO3 as additional means of removing atmospheric CO2. Important factors to increase the rate of CO2 bioremediation such as initial cell concentration, input CO2 concentration, and aeration rate are reviewed and discussed.

7.1 Introduction

In 2012, 34.5 billion tons of CO2 were emitted through human activities, and in 2013, an unprecedented modern age atmospheric CO2 concentration of more than 400 ppm was measured (Olivier et al. 2013). Carbon capture and sequestration (CCS) offers an effective solution to mitigate environmental impacts and can be

M. J. Raeesossadati • H. Ahmadzadeh (H)

Department of Chemistry, Ferdowsi University of Mashhad, Mashhad

1436-91779, Iran

e-mail: h. ahmadzadeh@um. ac. ir

M. P. McHenry

School of Engineering and Information Technology, Murdoch University, Murdoch, WA 6150, Australia

N. R. Moheimani

Algae R&D Centre, School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia

© Springer International Publishing Switzerland 2015

N. R. Moheimani et al. (eds.), Biomass and Biofuels from Microalgae,

Biofuel and Biorefinery Technologies 2, DOI 10.1007/978-3-319-16640-7_7 considered a long-term remediation policy (Yang et al. 2008). There are a number of CO2 remediation methods that can be classified in three main categories: capture, separation, and fixation.

Power plant CO2 capture can be divided into several scenarios, such as post­combustion process, pre-combustion, and oxy-combustion (Fig. 7.1) (Figueroa et al. 2008; Yang et al. 2008), and being stored in aquifers, porous geologic depleted oil and reservoirs, and deep ocean floors. In post-combustion processes, CO2 is separated from other flue gas constituents. In pre-combustion capture, carbon is removed from the fuel before combustion, and in oxy-combustion, the fuel is burned in an oxygen stream that contains little or no nitrogen (Figueroa et al.

2008) . Furthermore, other chemical approaches, such as amine absorption, ammonium absorption, molecular sieve adsorbent, and adsorption by activated carbon (Bezerra et al. 2011; Thote et al. 2010), are amenable for CO2 separation (Yang et al. 2008). The disadvantages of these methods are the use of large amounts of absorbents and solvents which makes the processes generally expensive (Figueroa et al. 2008), in addition to the processes relatively undeveloped and the possible wider impacts of the use of these chemicals which are not well understood (Wang et al. 2008).

In contrast to traditional methods of carbon capture, biological remediation processes via photosynthesis are major contributors to atmospheric CO2 remedia­tion (approximately 12 billion tons per year) (Bilanovic et al. 2009), with photo­synthetic organisms in the oceans responsible for removing over 40 % of annual

CO2 emissions (Pires et al. 2012). Bioremediation of CO2 can be accomplished through forestation, ocean, fungi, cyanobacteria, and algae (Skjanes et al. 2007) (Fig. 7.1). It is estimated that 1.4 ± 0.7 Gt carbon is captured by terrestrial systems from atmosphere via photosynthesis (Yang et al. 2008). The oceans store more CO2 than terrestrial vegetation (Israelsson et al. 2010), with around 38,000 Gt carbon, and about 1.7 ± 0.5 Gt taken up annually from the atmosphere (Yang et al. 2008). The production of phytoplankton at 50-100 Gt carbon annually is much higher than that of terrestrial vegetation. While part of the carbon is released back into the atmosphere by respiration, a large fraction would descend into the deeper ocean in the form of particulate organic matter either by the death of phytoplankton or after grazing. This sequestration process could be enhanced by ocean fertilization that refers to the practice of increasing limiting nutrients to stimulate the production of phytoplankton (Yang et al. 2008).

Organisms that can convert CO2 into organic molecules are called autotrophs and include plants, algae, some bacteria, and some archaea. Microalgae are the most promising bioremediation alternative for many sources of CO2 emissions. They have the capability of removing 10-50 times more CO2 than terrestrial plants, primarily due to more chlorophyll per unit area (Raeesossadati et al. 2014). Mic­roalgae can also utilize CO2 from different sources, such as atmospheric CO2, industrial exhaust gases, or CO2 in the form of soluble carbonates (e. g., NaHCO3 and Na2CO3) (Kumar et al. 2010). Open ponds and closed photobioreactors (PBRs) are commonly used for culturing microalgae to both consume CO2 and produce useful products (Kumar et al. 2010; Pires et al. 2012). Many microalgal strains can tolerate extreme environments and are able to grow with high production rates in large open ponds (e. g., Dunaliella, Spirulina, and Chlorella sp.), whereas closed PBRs allow better control of cultivation and reduce contamination issues (Eberly and Ely 2012; Pires et al. 2012). Despite many advantages of closed PBRs, large — scale open ponds are usually used for commercial microalgae production due to lower investment and production costs per unit of output (Lee 2001; Posten 2009). The ability of microalgae to bioremediate atmospheric CO2 is commonly thought of as dependent on freshwater and land availability, and the associated concerns of negatively influencing food security (Borines et al. 2011a, b; Clarens et al. 2010; McHenry 2012; Moheimani et al. 2013). However, marine and hypersaline mic­roalgae (eukaryotic or prokaryotic) can fix CO2 with almost no need for freshwater (McHenry 2010, 2013; Moheimani et al. 2012a; Sing et al. 2013). As such, microalgae are now one of the most promising alternatives to bioremediate many sources of CO2 emissions (de Godos et al. 2010). The authors have selected the term “bioremediation” as we are discussing temporary fixation of CO2 in the microalgal biomass. Furthermore, biomass productivity plays a significant role in any microalgae production system, and the production of many target constituents is dependent on primary biomass productivity (including the production of lipids, hydrocarbons, polysaccharides, and other energy storage compounds). The more biomass productivity in any microalgal system is the results of more photosynthetic fixation of CO2. Therefore, to produce industry-scale microalgae biomass, there is a need for cheap carbon source and nutrients.