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).

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.

Network refinement (Fig. 10.3c) can be viewed as reconciliation between the content of the model and the available biochemical and genomic data, with the end result of enhancing the reconstructed network. This reconciliation is done based on agreements of model simulations and updated genomics, physiological, and bio­chemical knowledge. A crucial step in the reconstruction of genome-scale meta­bolic models is filling the gaps to decrease the number of dead-end metabolites and improve network connectivity. Metabolic network gaps are filled by the addition of reactions that are missing in the network yet have corroborating evidence for their existence in the system. These may include spontaneous reactions that are not associated with gene products as well as extracellular and intracellular transport reactions and exchange reactions.

Models may not predict the production of biological compounds with existing biochemical evidence if the prerequisite genes have not been added to the model. Manichaikul et al. (2009), using Chlamydomonas as a model, described how genomic data can be used to fill gaps in metabolic models. In their approach, not only genomics and other experimental evidence contributed to the refinement of the network, but also the model itself informed “genomics,” of the presence of missing annotations, justifying the use of more sensitive sequence search and annotation tools to recover the missing genes. One example that can illustrate this is lactate dehydrogenase (LDH), which initially was absent from the Chlamydomonas gene annotation, yet the model reconstruction showed the need for the LDH enzyme in the Chlamydomonas pyruvate metabolism pathway. A PSI-BLAST analysis was carried out to identify the gene encoding LDH; the gene was subsequently added to the model. Additionally, orphan genes, or those biochemically characterized metabolic enzymes lacking sequence data, can be assigned GPRs by reviewing metagenomic sequence data to provide sequences for the missing enzymes. This approach has been experimentally validated (Yamada et al. 2012).

Algenol (Florida, USA) has been continually refining a process for continuous growth and harvest of ethanol excreted by modified cyanobacteria. In this process, ethanol is released by the organism in the vapour phase and then captured for extraction using a novel distillation process, eventually the spent microalgae bio­mass is converted to fuel using a variant of the HTL process. Algenol claims the following figures for their pilot plant (http://www. algenol. com/):

• Yield: 8000 gallons per acre of total liquid fuel production (80,000 L/ha) of which 85 % is ethanol and the remaining 15 % is hydrocarbons

• Cost: $1.27 per gallon

Two other companies pursuing milking-based projects are Joule unlimited and Proterro who are focused on chemicals/fuels and sugars as feedstock to traditional biofuel processes, respectively. In all of these approaches, the process is funda­mentally different as the milking process extracts the oils, ethanols or other chemicals of interest from the growth medium without killing the microalgae. As a comparison, the traditional microalgae production systems ‘kill’ the ‘cow’ (microalgae) to extract the ‘milk’ (oil) rather than keeping the cow (microalgae) productive and continually harvesting the milk (oil) (Moheimani et al. 2013a). Milking addresses the shortfalls of the existing production systems in two major ways:

• Nutrients—Only the products of interest are removed (which typically contain very low N and P), and as a result, there is a limited requirement for fertilisers. Only water, CO2 and sunlight are required to continually produce the compounds

• Dewatering—Microalgae are typically not removed from the culture to be milked to limit the need for dewatering (Moheimani et al. 2013b).

These systems are currently at various stages of early development. Despite this, the potential of these novel approaches to address the major issues with traditional methods warrants their continued investigation.

Many industries and human activities generate wastewater, and as a consequence, there are many different types of wastewaters, each with a different chemical composition and volumetric production over time. Table 5.1 gives an overview of different types of wastewaters and their content of N and P. The two major sources of wastewater are domestic wastewater and wastewater derived from animal manure. Each person supplies about 3 kg N per year through domestic wastewater, which translates to about 21 million ton of N per year for a global population of 7 billion (similar to the estimate of Smil 2002; Van Harmelen and Oonk 2006). The major livestock animals that produce manure are pigs, cattle, and chickens.

Chickens produce a relatively dry type of manure that is suitable for application as fertilizer on agricultural land and we do not consider the N supply from chicken manure for algae production. Pigs produce about 16 kg N animal 1 year 1, cattle 35 kg N animal-1 year-1, and dairy cattle 75 kg animal-1 year-1. With global population sizes of 1 billion pigs, 0.9 billion cattle and 0.25 billion dairy cattle, wastewater from pig and cattle manure can theoretically provide 65 million tons of N, or about 3 times more than domestic wastewater (similar to the estimate of Van Harmelen and Oonk 2006). If we assume a “N” content of microalgal biomass of 7 %, the total human, pig, and cattle wastewater N nutrient is enough to produce about 778 million ton dry microalgal biomass per year. This is in the same order of magnitude as the global production of wheat or of corn. Although this is a lot of biomass, it can produce only about 233 million ton of oil (assuming a 30 % lipid content in microalgae). This corresponds to 1800 million barrels of oil, or only about 5 % of the global oil consumption. Thus, wastewater alone cannot supply sufficient nutrients for microalgal biomass to make a large contribution to the world’s energy demand. This conclusion is in line with Peccia et al. (2013) or Chisti (2013), who estimated that nutrients from domestic wastewater of a typical large city can only produce enough microalgal biofuel to supply 3 % of the fuel demand of that city. If microalgal biofuels are ever to make a larger contribution to the global fuel demand, it will be essential to recycle the nutrients during conversion of microalgal biomass to biofuels (Venteris et al. 2014). Although animal manure is a potentially significant source of nutrients for microalgae production, it should be noted that a significant proportion of animal manure is already used as a fertilizer in conventional agricultural production (Bouwman and Van Der Hoek 1997). Because synthetic fertilizer prices are increasing, the value of nutrients in animal manure also increases. Therefore, the use of animal manure as fertilizer in conventional agriculture is likely to increase in the future (Shilton et al. 2012), and microalgae and conventional agriculture may compete for animal manure nutrients. However, the main limitation of the use of raw animal manure in conventional agriculture is the high transport cost resulting from the high water content of animal manure. In areas where livestock numbers are high and agricultural crop production is nutrient — limited, it will unlikely be economically feasible to transport animal manure to the field, and microalgae may become a more attractive option to process large volumes of manure on a relatively small land area.

In addition to manure, there are many other sources of wastewater that could be used for microalgae biomass production, such as wastewater from olive mills, wineries, breweries, vegetable processing, tanneries, or the paper industry (Cai et al.

2013) . Some emerging technologies also generate a lot of wastewater. The use of anaerobic digestion to convert organic waste streams into methane is growing worldwide and generates a nutrient-rich effluent that could be processed with microalgae (Uggetti et al. 2014). Aquaculture is also increasing worldwide and generates a similar nutrient-rich wastewater that may be suitable for treatment using microalgae (e. g., Van Den Hende et al. 2014). The volumes that are produced by these industries are relatively small compared to the volumes of domestic and animal manure wastewater. Nevertheless, microalgae may be a solution to treat some of these wastewaters as conventional water treatment technologies may be too expensive or ineffective.

The RNAi pathway has been studied in the unicellular green alga C. reinhardtii, and used as a reverse genetics tool in different algal species. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have been identified in four algal species, C. reinhardtii (Molnar et al. 2007; Zhao et al. 2007), Porphyra yezoensis (Liang et al. 2010), Phaeodactylum tricornutum (De Riso et al. 2009), and Ectocarpus siliculosus (Cock et al. 2010). However, RNAi mechanisms and their applications remain largely uncharacterized in most algae. RNAi against specific genes can be induced by the introduction of exogenously synthesized dsRNAs or siRNAs into cells or whole organisms (Cerutti et al. 2011; Moellering and Benning 2010; Molnar et al. 2009). Within algal species, in vitro — synthesized long dsRNAs have been electroporated into Euglena gracilis cells and shown to silence two endogenous genes homologous to the introduced dsRNAs (Iseki et al. 2002; Ishikawa et al. 2008). Recently, an RNAi triple knockdown of the three most abundant LHCII proteins (LHCBM1, 2 and 3) has been reported in Chlamydomonas with the aim of increasing the efficiency of photobiological H2 production (Oey et al. 2013). Artificial microRNA (amiRNA) expression success­fully exploits endogenous miRNA precursors to generate small RNAs that direct gene silencing in C. reinhardtii (Molnar et al. 2009; Schmollinger et al. 2010; Zhao et al. 2008). Zhao et al. (2009) developed an artificial amiRNA-based strategy to

knock down gene expression in Chlamydomonas using an endogenous Chlamydo — monas miRNA precursor (pre-miR1162) as the backbone. Other studies show that amiRNAs can be used as a highly specific, high-throughput silencing system, and they propose that they will become the system of choice for analysis of gene function in Chlamydomonas and related organisms (Molnar et al. 2009; Schmollinger et al.

2010) . The synthesized miRNAs provide a convenient tool for reverse genetic studies in Chlamydomonas. More recently, epitope-tagged protein-based amiRNA (ETPamir) screens were developed, in which target mRNAs encoding epitope — tagged proteins were constitutively or inducibly co-expressed in protoplasts with amiRNA candidates targeting single or multiple genes (Li et al. 2013). This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA

Fig. 8.2 Genome engineering tools. a miRNA pathway. MIR genes are transcribed by RNA polymerase II into pri-miRNA transcripts that are further processed into pre-miRNAs harboring a characteristic hairpin structure. From the stem of the pre-miRNA the miRNA/miRNA* duplex is excised by DCL1 and can be assisted by HYL and SE proteins. miRNA-guided AGO-containing RNA-induced silencing complex (RISC) directs mRNA cleavage or translation inhibition of the target transcript. b Summary of Transcription activator-like effectors (TALEs) nuclease. Custom — designed nucleases introduce double-strand breaks with high precision at predetermined genomic loci. Double-strand breaks are either repaired by error-prone non-homologous end-joining (NHEJ) or high fidelity homologous recombination (HR). NHEJ repair causes random insertions and/or deletions of nucleotides around the target site and some of these mutations will knockout gene function. Gene replacement, tagging, or correction can be achieved by HR-mediated targeted integration of a donor construct that is provided together with a nuclease pair. c CRISPR/Cas9 mediated target DNA cleavage. The CRISPR loci include Cas genes, a leader sequence, and several spacer sequences derived from engineered or foreign DNA that are separated by short direct repeat sequences. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3′ end of the target sequence and is followed by DNA repair by the endogenous cellular repair machinery expression/processing and antibody unavailability. These screens could improve algal biofuel engineering research by making amiRNA a more predictable and manageable genetic and functional genomic technology. From a practical perspec­tive, RNAi is becoming a customary method for directed gene silencing in algae. As the necessary molecular tools are developed, RNAi approaches are expected to contribute to the functional characterization of novel genes, as well as to the strain engineering of algae (Fig. 8.2a). Ultimately, RNAi technology may provide much-needed insights into gene function, metabolic pathways, and regulatory networks allowing us to comprehend the role of algal species in nature, as well as to engineer these organisms for the synthesis of valuable bioproducts.

Biodiesel is a diesel fuel consisting of mono-alkyl esters of long-chain fatty acids that are generally made by the transesterification of lipids in animal fat or vegetable oils such as soybean, sunflower, rapeseed, and oil palm (Hankamer et al. 2007; Li et al. 2007; Ma and Hanna 1999). As an alternative, microalgae have become popular for the renewable generation of hydrocarbon-based biofuels with high biofuel yields relative to those from plants (Eroglu and Melis 2009; Li et al. 2007).

Immobilization of the hydrocarbon-rich microalgae, Botryococcus braunii and Botryococcus protuberans, in alginate beads yielded a significant increase in the chlorophyll, carotenoids, cellular growth, and lipid contents of the cells during their stationary growth phase (Singh 2003). Bailliez et al. (1985) also observed enhanced hydrocarbon production for the B. braunii cells immobilized in calcium alginate gel as a result of enhanced photosynthetic activity.

In a study by de-Bashan et al. (2002a), C. vulgaris and C. sorokiniana micro­algal cells were individually co-immobilized with A. brasilense growth-promoting bacterium in alginate beads. They found that the presence of the growth-promoting bacterium within the immobilization matrix significantly enhanced the metabolism of Chlorella strains and yielded higher lipid and fatty acid production.

Li et al. (2007) used immobilization technology for the transesterification of algal oils, using immobilized lipase enzyme from Candidia sp. Initially, they grew Chlorella protothecoides cells in large-scale photobioreactors at three different sizes (5, 750, 11,000 L) yielding high lipid contents in the range 44-49 % per dry cell weight. Then, immobilized lipase enzyme from Candidia sp. was used to catalyze the transesterification of the lipids from C. protothecoides, yielding biodiesel production rates of 7.02, 6.12, and 6.24 g L-1 from 5, 750, and 11,000 L biore­actors, respectively. They also highlighted that the quality of this Chlorella biodiesel was comparable to that for conventional diesel fuels (Li et al. 2007).

For microalgae biofuel production, a major cost factor is the provision of water and nutrients (Davis and Aden 2011; Borowitzka and Moheimani 2013), which can both be provided by wastewater. Microalgae ponding systems were developed in the 1950s for municipal sewage treatment (see Oswald 2003 for a review of the early work), and this approach continues to serve as a starting point for the development of cost-efficient algae biomass for fuels production. At least 70 % of the cost of wastewater treatment can be attributed to secondary and tertiary treat­ment. Much of this is due to the energy costs of oxygen transfer in biological secondary treatment and chemical requirements in tertiary treatment.

Dr. William Oswald at the University of California-Berkeley and his colleagues over the following 50 years (Oswald 2003) developed the fundamental engineering design parameters and described the basic biological processes in bioremediation in high-rate ponds. Microscopic algae convert about 2 % of total solar energy to algal biomass. The photosynthetically generated oxygen is consumed by bacterial pop­ulations that decompose organic wastes to simple nutrients including CO2. Although algae-based secondary and tertiary treatment is economically feasible, at least in warm regions with ample land, few municipal algae ponds attempt to control species composition or even harvest the algal biomass (Benemann and Oswald 1996). Two of the persistent problems noted in the early years of inves­tigation were maintaining a stable algal population in the treatment ponds and harvesting the algae in an efficient and economic manner. Open pond cultures are subject to all the variations that occur in natural ponds and lakes. At any given time, there can be a major shift in algal dominance such as a transition from green algae to cyanobacteria (potentially toxic) or there can be a sudden collapse of the com­munity structure due to algae bloom crashes and predation by zooplankton. The sudden death of phytoplankton communities, or algal blooms, is thought to result from several factors including insufficient light for photosynthesis, limiting nutri­ents, phycoviruses (Brussaard 2004), the aging of the blooms, and perhaps by photoinhibition in gas-vaculate cyanobacteria.

Successful open-air algal monoculture is currently limited to a small number of species that can tolerate extremes of pH or salinity that preclude invasion by competing microbes and undesired predators conquering unenclosed outdoor ponds. Enclosed bioreactors mitigate some of the problems of maintaining mono­cultures and predation issues, but capital and labor costs limit their use to pro­duction of high-value products. Other major hurdles to economically feasible algae — based biofuels include the use of high oil strains adapted to the local environment, development of resource-specific production and management systems, and, at least in the short term, coupling algae culture with mitigation of environmental problems and co-production of high-value compounds.

Minimal nutritional requirements for algal growth can be estimated from the approximate molecular formula of algal biomass: C(048) H(183) N(011) P(0.0i) (Grobbelaar 2004). The chemical composition of municipal and dairy wastewaters typically has less N than P relative to algal biomass (Fulton 2009). Although CO2 limits algal growth in high-rate oxidation ponds (Benemann and Oswald 1996), when CO2 is supplemented, N typically limits algal growth on municipal (Bene — mann et al. 1977) and agricultural (Lundquist et al. 2011) wastewaters. N limitation has long been known to be a trigger for lipid synthesis in some algal species. In a variety of microalgae, as the nitrogen or phosphorus levels drop limiting growth, there is a rapid increase in oil (lipid) content (Takagi et al. 2000; Li et al. 2008). The mechanisms involved in the “N-trigger” have remained elusive. Although recently comparative proteomics and transcriptome analysis of the haptophyte Tisochrysis lutea (formerly known as Isochrysis galbana) have revealed a wide variety of proteins and transcripts involved in various pathways including lipid, carbohydrate, amino acid, energy, and pigment metabolisms, photosynthesis, protein translation, stress responses, and cell division in strains are subjected to N limitation (Garnier et al. 2014). Most of the oleaginous algae are non-motile. They are thought to produce lipids as a buoyancy compensator to position them in the ideal part of the water column during the light/dark cycle of photosynthesis. As a survival mecha­nism to counter the depletion of nutrient supplies, they may produce high levels of lipid to bring them to the surface where the wind may blow them to a more nutrient — rich environment. This insight favors the concept of two-phase semi-continuous cultures for wastewater treatment to promote lipid biosynthesis. As the rapidly growing algae take up all the available nitrogen and phosphorus, biosynthetic pathways for growth are nutrient limited and oil content rises.

Figure 6.1 is a flow diagram of a hypothetical algae-based wastewater treatment systems with multiple benefits that take into account the drawbacks described in previous algae-culturing systems. The treatment process is based on a sequence of events designed to yield a high-quality effluent and a consistent supply of algae biomass:

1. Filtered primary wastewater is added to the first of three shallow raceways. There are three raceways: one is filling, one is in the algae growth stage, and the third raceway is in the harvesting stage. A fourth raceway may be needed to account for variations in productivity.

2. The filtered primary effluent is rich in biochemical oxygen demand (BOD), ammonium, and organically bound phosphorus. This effluent is mixed with a high concentration of a pure strain or strains of algae that are grown in a bank of photobioreactors. The algae in the photobioreactors are added to the pond when they are at or near the top of their log-phase growth. The dilution of the concentrated algae into the pond is adjusted to the lower end of early log-phase growth. These ideal strains were selected for their ability to grow rapidly on wastewater, adapted to local waters, high lipid content, and harvestability. In the daylight, the algae produce large quantities of oxygen that facilitate the oxidation of the various organic compounds that contribute to the BOD. During the dark cycle of photosynthesis, the algae can take up a variety of organic compounds including the organic compounds that were degraded during the light cycle of photosynthesis.

Possible Extraction of

Commercially Important

Feedstock for Polymer

be composted to produce a fertilizer and the M Pr°duction or ioplastics

high-nutrient liquid can be used

Fig. 6.1 Flow diagram of an algae-based wastewater treatment system. The boxes with the dashed border indicate an end product or benefit

3. Due to the log-phase growth, in 72-120 h, the algae biomass has increased fourfold to eightfold; the nutrients have been assimilated by the algae, and the BOD has dropped to the level associated with tertiary treatment. The short residence time in the open ponds lowers the likelihood of contamination by another strain of algae or predation by zooplankton mainly by competitive exclusion (Lang 1974; Hillebrand 2011).

4. The algae can be harvested by one of many physical separation processes (for more information of dewatering, see Chaps. 12—14), and the tertiary effluent can be disinfected and prepared for some form of water reuse. At this point, the algae can be ground up to yield a green crude or lysed in order to separate the lipid content from the aqueous and solid fractions.

5. The green crude can be processed into a variety of fuels in the same way the crude oil is processed. If the cells were lysed and just the lipid fraction was isolated, it would then be converted to biodiesel by one of a variety of physical, chemical, or enzymatic processes.

6. The algae biomass could potentially contain a number of valuable phyto­chemicals that can be extracted and purified for use in the chemical or personal care products industry. The biomass could also be used as a feedstock for the production of synthetic polymers.

7. If the previous option is not feasible, the cracked algae cells can be put into a digester to produce methane. Unlike activated sludge, this organic matter has a uniform composition, and since the cells are already lysed, the degradation of the organic matter and subsequent production of methane should be more efficient than digesters filled with activated sludge.

8. The energy demands on this process should be considerably less than tradi­tional secondary and tertiary treatment such that the energy produced by cogeneration could be sold on the grid.

9. The CO2 that is produced by burning the methane is usually released into the air. In order to raise algae in high concentrations, it is necessary to add CO2 to the water. By bubbling in CO2 into the rapidly growing algal cultures, it is possible to capture the majority of CO2 produced by the cogeneration system (for a recent review, see Raeesossadati et al. 2014 and Chap. 7).

10. In the event that carbon “cap and trade” rules are implemented, this process provides a way for substantially shrinking the carbon footprint of a wastewater treatment plant.

11. The solid residue from the digester can be composted to produce a high-quality fertilizer, and the liquid fraction of the digester (mixed liquor) can be sterilized and used as a nutrient source for the bank of photobioreactors.