Commercial microalgae biofuel production is dependent on the unique microalgal species chosen, the management of biotic and abiotic conditions, production costs (energy, nutrients, water, land, chemicals, etc.), co-located industrial facilities, and downstream applications and markets (Borowitzka 1999; Kunjapur and Eldridge 2010; McHenry 2010, 2013). Because the concentration of microalgae in open pond systems is so low [usually between 0.5 and 2 g/L (Fon Sing et al. 2011)], dewatering and harvesting/processing will heavily influence the evolution of upstream microalgae production, strain selection, water/nutrient recycling tech­nology, and other processes (Moheimani et al. 2013). Primary dewatering is typi­cally achieved through flocculation, followed by separation from the water via settling or floatation (Moheimani et al. 2013). Some microalgae naturally flocculate, while others respond to chemical flocculants or non-chemical methods (Brady et al. 2014; Moheimani et al. 2013; Sukenik and Shelef 1984). For example, Origin Oil and Diversified Technology have used pulsed electric fields to enhance flocculation and lyse microalgae (Moheimani et al. 2013). During post-primary dewatering, the remaining water in microalgae pastes is usually removed by heating, which if not using low grade or waste heat can also include a high-energy burden (McHenry 2013; Moheimani et al. 2013). Secondary dewatering or mechanical drying gen­erally incudes physical separation of the water and the microalgae using centrifuges or decanters that are often energy intensive and relatively inefficient (Moheimani and McHenry 2013; Moheimani et al. 2013; Solix Biofuels 2011b). However, this area of research is a fast-moving field with some companies including Evodos achieving mechanical centrifuge electricity consumption reductions when dewa­tering water and microalgae to a paste (Moheimani et al. 2013). Evodos centrifuges exhibit practically zero cell shearing, thermal damage, without chemicals and collecting minimal bacteria requiring only <1 kWh/m, and 5.5 kWh/kg DW (Evodos 2011). Nonetheless, for industrial-scale volumes of water, this energy consumption per unit of output remains prohibitively high.

There are numerous competing approaches focussed on reducing dewatering energy demands in microalgae production (McHenry 2013). For example, General Atomics, a long-time defence contracting company based in California, is evaluating Algaeventure Systems microalgae “harvesting, dewatering, and drying” (HDD) technology with the ability to reduce dewatering energy consumption considerably using unique conveyer centrifugal technology (Li 2012; Moheimani et al. 2013). Algaeventure Systems Inc. is a spin-off company from Univenture, Inc., which is a plastic packaging manufacturer, a large-potential high-value microalgae-based co­product potentially cross-subsidising biofuel production (Moheimani et al. 2013). Taking another unique method to dewater and process biofuel precursors is the company Algenol, which has developed a unique DIRECT TO ETHANOL® process using a 15-m-long and 1.5-m-wide semi-transparent polyethylene film outdoor photobioreactor containing treated sea water, microalgae, nutrients, with a volume of air above the water. Algenol uses a “hybrid” microalgae that reportedly produce ethanol intracellularly which diffuses through the cell wall into the culture medium and evaporates along with water into the internal air volume. The water/ethanol solution condenses on the inner surface of the photobioreactor and is collected, concentrated, and distilled (Moheimani and McHenry 2013). Algenol states that their DIRECT TO ETHANOL® process produces around equal amounts of freshwater and ethanol, with collaborators membrane technology research (MTR) using their Bio — Sep™ technology—a membrane distillation technology. Similarly, Solix’s from Colorado has developed a “LumianTM AGSTM” photobioreactor which is a series of water-filled metal tanks supporting semi-submerged transparent microalgae cultures circulated by weighted rollers, with independently controlled air and CO2 from a co­located coal-bed methane production facility (Moheimani and McHenry 2013; Solix Biofuels 2011a). The twenty 36-m-long 200-L bags sum to a total culture capacity of around 4 kL (Solix Biofuels 2011c). Solix’s Colorado Coyote demonstration facility can produce a maximum of 28 kL/ha/year of microalgal oil, with culture peak yield rates equivalent to around 19 kL of oil/ha/year, or around 5 g of oil per m2 per day (Moheimani and McHenry 2013). However, Solix is primarily a culturing technology provider and has not focussed on downstream energy and dewatering technology.

Another unique production approach is underway in New Zealand, where a private company, Aquaflow, is specialising in harvesting wild microalgae from municipal sewage ponds and high-nutrient waters. Aquaflow solely relies on water remediation and equipment sales rather than microalgae as their business model. In 2008, Aquaflow partnered with Honeywell UOP to use UOP/Eni Ecofining™ and the Canadian company Ensyn’s rapid thermal processing (RTP™) fast biomass pyrolysis (*75 % bio-oil output by volume) to provide liquids for Honeywell’s Green Die­sel™ and Honeywell Green Jet™ fuel production (Ensyn 2011), using hydropro­cessing to produce catalysts and thermal energy for output separation (Moheimani et al. 2013; Regalbuto 2011). Aquaflow currently operate with around 60 ha of open mixed sewage and municipal and agro-industrial waste oxidation ponds (serving a population of 27,000 and water flows of 5 GL/year.) The ponds are continuously harvested using systems built inside a 40 ft sea container using dissolved air flotation and polyelectrolyte flocculation (70-90 % recovery), followed by a belt press for extraction, processing wastewaters at a rate of 35 m3/h to produce a wild microalgae liquid concentrate at 8-10 % microalgae by volume (Moheimani et al. 2013).

To avoid the associated high energy and material costs of dewatering, integrated microalgal biorefineries have received much attention in the literature (Chisti 2007; Wyman and Goodman 1993), and recent approaches have focussed on hydrothermal liquefaction of relatively low-lipid biomass from mixed microalgae species to pro­duce a crude oil replacement in the presence of water at medium temperatures and pressures (Biller and Ross 2011). de Boer et al. (2012) showed that in situ hydrolysis and esterification of wet biomass and hydrothermal liquefaction was the most ener­getically feasible process in a comparison of four of the most promising methods to convert microalgae into biodiesel, including pulsed electric field-assisted extraction, transesterification, and in situ acid catalysed esterification of dry biomass. However, the present fundamental diversity of approaches for microalgae dewatering and the associated energy efficiency options demonstrate that microalgae dewatering is far from a mature field on the verge of commercialisation. This research discusses an alternative approach that is a potential tool for achieving low-cost primary dewatering of microalgae for economic biofuel production using inexpensive “chemical-free” approach to autoflocculation that is appropriate to large-scale industrial production systems using existing bulk agricultural commodities.

The power output under constrained lighting situations from both a crystalline and amorphous silicon solar module has been calculated using the model outlined earlier. This is independent of the species of algae used as the power produced by the photovoltaics relies only on the portion of the spectrum they would receive. The threshold determines the portions of the spectrum provided to the photovoltaics as shown in Fig. 15.5. The power produced is not zero when the entire PAR is provided to the microalgae as there is an extensive part of the spectrum beyond PAR which photovoltaic devices can convert to electricity. It is clear that crystalline silicon solar cells are much more efficient in the regions outside of PAR than amorphous silicon is. This is due to the extended spectral response of crystalline silicon into the infrared part of the spectrum and the higher efficiencies of the crystalline solar cells (Fig. 15.3). The energy generated by these crystalline silicon solar cells can be used to power additional lighting to add more irradiance to the microalgae. If a LED system with external quantum efficiencies in the order of 55 % (Krames et al. 1999b) is used, a substantial amount of additional illumination can be provided to the microalgae.

The model we have created for this scenario is highly dependent on the absorption spectra of the microalgae. The power generated by the PV cells can be directed to a series of LEDs which will provide additional illumination to the microalgae. As can be seen in Fig. 15.6, the augmented power absorbed by the

microalgae is distinctly higher from some species when compared to others. Notably, this is chaetoceros and tetraselmis. The reason for this is apparent in Fig. 15.2. These two species of microalgae have a much greater absorbance than the other species in the measured absorbance data. This does not necessarily mean they are the most productive and only that they absorb the greatest portion of irradiance. The second trend which appears in the graphs in Fig. 15.6 is that a system aug­mented with electricity generated by crystalline silicon solar cells will generate more power and have a larger portion able to be provided back to the microalgae via LEDs. This highlights the importance of a highly efficient collecting device. For the remainder of this work, we will focus on results from the crystalline silicon parts of the model which are a best-case scenario.

A more useful visualization of this data is to examine the change in the amount of power absorbed by the microalgae as the threshold is changed. Figure 15.7 shows the change in power absorbed by various microalgae species when compared to the minimum situation. That is, the situation where all irradiance between 400 and 700 nm is transmitted through to the microalgae. Additional illumination is still provided by LEDs using the IR and UV parts of the spectrum. When compared to this baseline, it can be seen that even if all the irradiance in PAR is transmitted to the algae, there is a boost of 20 % in the total amount of power absorbed by the microalgae. This is from the additional irradiance provided by the LEDs which are powered by infrared radiation (>700 nm) captured by the crystalline silicon photovoltaics. As the threshold is increased, the total amount of power provided to the microalgae decreases and beyond 50 %, there would be no net benefit for this system. At first glance, this would seem to indicate there is not a great deal of advantage in filtering the light as described in this model and combining electricity and biofuel production. However, it needs to be recognized that much of the power being absorbed by the microalgae may not be assisting in photosynthesis. There is

still significant absorption in other portions of the spectrum. If, for example, the green portion of the spectrum is not required for photosynthesis, it can be entirely removed from the spectrum provided to the microalgae and instead converted to blue or red light which will be absorbed efficiently by the microalgae.

A better comparison would be to the 50 % threshold. This equates to a full-width half-maximum band-pass around the main absorption peaks of the microalgae as seen in Fig. 15.2. This situation is shown in Fig. 15.8. From this figure, it can be seen that there are gains in the amount of irradiance absorbed by the microalgae by

in excess of 100 % in some situations and for some species. This is a significant increase in irradiance absorbed by the microalgae and should lead to more pro­ductive growth.

A combination of these two energy production methods (solar energy and chemical energy) can efficiently use the whole solar spectrum. We recognize that an area covered by PV panels of the same scale as a microalgae farm would produce more electrical energy than the algae can store as chemical energy. However, the advantage of our proposed method is the production of chemical energy for transportation or other high value crops and can increase the productivity of mic­roalgae systems.

This suggests that a combination of the two energy production systems would allow for a full utilization of the solar spectrum allowing both biofuel and electricity production from the one facility. This makes efficient use of available land, or it can enhance biofuel production by management of the spectrum and the addition of targeted illumination. Therefore, we propose a co-production system that uses an active filter or photovoltaic system above a microalgae pond to capture and effi­ciently convert the whole solar spectrum into usable energy or products. While the mechanism for splitting the spectrum is not fully determined as yet, there are several candidate options, including a specifically tailored semitransparent thin-film PV, luminescent solar concentrators, or other advanced energy harvesting flat glass panel that match the spectrum not used by the microalgae. One excellent candidate technology system that can transmit arbitrary visible light wavebands, capture the infrared part of the spectrum, concentrate it on the edge of a glass panel, and convert it to electricity has been recently developed and patented (Rosenberg et al.

2013) .

Karne de Boer and Parisa A. Bahri

Abstract Microalgae biofuels have been under development for the last 40 years; however, in the last 6 years, this development has intensified due to higher oil prices and wider acceptance of anthropogenic climate change. Despite the excellent potential of algal biofuels, they are not yet commercially viable. The reason for this lack of progress is examined in this chapter by firstly reviewing the range of different technology options for biofuels from microalgae. Secondly, an analysis of the available techno-economic and energy assessments is performed highlighting the effect that each system element has on the overall viability.

17.1 Introduction

Microalgae are a feedstock of great interest for the production of energy, fuels, food, high-value nutritional supplements and specialty chemicals. Microalgae attract this attention because they have high photosynthetic efficiency, can be grown at massive scale on non-arable land, grow rapidly, can thrive in salt or brackish water and naturally produce a range of compounds with commercial value (Borowitzka and Moheimani 2010). Due to the high potential of fuel production from microalgae, research and investment into microalgae development typically occur in earnest after oil price spikes, e. g. the aquatic species program in the USA after the 1970s oil embargo (Sheehan et al. 1998) and the rush of new algae companies and research after the rapidly increasing oil prices between 2007 and 2012 (Ribeiro and da Silva 2012).

Despite these promising characteristics, no one has been able to commercially cultivate and process microalgae at scale for the purpose of producing fuels. The central reason for this slow progress is that it is not economically viable

K. de Boer (H) • P. A. Bahri

School of Engineering and Information Technology, Murdoch University,

Murdoch, WA 6150, Australia

e-mail: karne@regenerateindustries. com © 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_17 (ANL et al. 2012; Benemann et al. 2012; DOE 2014). That is, it costs more to grow and convert the algae into fuel than the fuel is worth (Stephens et al. 2010).

This chapter reviews the existing techno-economic models and life cycle anal­yses (with a focus on energy consumption) that have been published on the large — scale production of algal biofuels. The purpose of this review is to provide readers with a clear grasp on future trends in microalgae economics and opportunities for improvement. This review is focused on the economic and energetic feasibility of the biofuel production process as these are the barriers to large-scale commercial deployment.

The main advantages of microalgae growth compared to land plants are the ability to grow on arid land using saline water (Fig. 1.1). This means that microalgae cultures will not compete with food crops over agricultural land and freshwater. However, microalgae, the same as any other photosynthetic organism, would still require fertilisers (especially nitrogen and phosphorous) to grow. If grown in sea water, macronutrients are necessary to be added to the culture to achieve high growth rate. Borowitzka and Moheimani (2013a) indicated that for producing

100,0 bbl of algal oil year 1, there is a need for 14,447 and 219 tons of nitrogen (as NaNO3) and phosphorous (as NaH2PO4), respectively. Such a high volume of fertilisers will significantly affect the overall cost of production. Furthermore, phosphorous is a non-renewable resource, and at current rates of extraction, global commercial phosphate rock reserves may be depleted in less than 100 years (Cordell et al. 2009). That means that algae cultures, irrespective of their product, will be in direct competition with food crops over fertilisers. Obviously, one very important consideration in developing any potential large-scale algae production facility is the recycling of the medium (Fig. 1.1). Recycling medium especially post-extraction/conversion would allow the recycling of a large amount of fertilisers especially if the wet biomass is being converted to biodiesel and biomethane (Fig. 1.1). Furthermore, there is a possibility of combining microalgae cultivation with wastewater treatment. Combining microalgae cultures with wastewater treat­ment plants (domestic or animal waste) can provide microalgae with required nutrients and result in lower cost wastewater treatment than traditional approaches.

The potential of combining microalgae cultures and domestic wastewater treatment was first proposed in 1960s with the main interest to produce biofuel (Oswald and Golueke 1960). There are currently some facilities around the world (i. e. New Zealand, USA) using high rate algal ponds (HRAPS) for treating tertiary domestic wastewater. In general, microalgae growth in tertiary-level wastewater treatment can significantly reduce the electromechanical cost of treatment (Craggs et al. 2013). Another advantage of using microalgae in the domestic wastewater treatment process is more efficient nutrient removal and sunlight-driven disinfection (Davies-Colley et al. 2005). Animal waste (i. e. piggery waste) can also be treated using microalgae cultures. The environmental impacts of intensive pig production can be significant. A poorly managed piggery may risk wastewater pollution to local waterways, produce odour emissions and release greenhouse gases into the atmosphere (Maraseni and Maroulis 2008). Wastewater generated through high- intensity pig production is high in ammonia and phosphorous while also having high chemical and biological oxygen demands (Olguin et al. 2003). High phos­phorous levels have been shown to correlate to high turbidity levels giving the effluent a dark colour (Ong et al. 2006). One wastewater treatment system that is gaining acceptance in Australian piggeries is anaerobic digestion ponds. These systems typically consist of a covered pond containing wastewater which is bio­logically treated by heterotrophic microorganisms in the absence of oxygen. The covered digesters allow the production and capture of biogas including methane and carbon dioxide. The benefits obtained from these ponds are the removal of solids through settling, capture of biogas for use as a biofuel and the reduction of odour emissions. The utilisation of methane as a fuel source can effectively reduce dependence on energy sources from outside the piggery. One challenge is that the anaerobic digestion effluent from piggeries is very high in ammonium (toxic to most organisms). If a process incorporating CO2 uptake such as algae culture was to be adopted, ideally CO2 (generated via burning CH4 or separated from the raw biogas stream) will be captured and reused within the piggery. A recent review of wastewater management in Australian piggeries recommended that along with anaerobic digestion, microalgae culture systems should be investigated further as a potential component of the Australian piggery wastewater management strategy (Buchanan et al. 2013).

To date, all trials on culturing microalgae on undiluted and untreated anaerobic digestion piggery effluent (ADPE) have failed to gain widespread acceptance in the industry. On the other hand, there are reports of the successful microalgal culti­vation on piggery anaerobic digestate after dilution with freshwater (Park et al.

2010) . Interestingly, in some cases, the digestate was diluted more than 15 times with freshwater. In the context of an Australian piggery system, such a method would never be practical due to the shortage of freshwater. Ayre (2013) isolated three microalgae capable of growing on undiluted, sand-filtered, piggery anaerobic digestate. This proof-of-concept study clearly illustrated the potential for culturing microalgae in such effluent with a high ammonium content. The produced algae biomass on piggery anaerobic digestate will sequester carbon and remove nutrients (i. e. nitrogen and phosphorous). The produced biomass could alternatively be used as pig feed, although the biomass pathogen load would need to be closely moni­tored (Buchanan et al. 2013). Another potential application for the biomass is the co-anaerobic digestion with the piggery waste.

Wastewater has a relatively low nutrient content, with usually less than 1 % N and less than 0.5 % P. Because of this low nutrient content, it is not cost-effective to transport wastewater over long distances to microalgal farms. Therefore, microalgal farms should be situated as close as possible to the wastewater sources. It is also costly to store wastewater during periods when microalgae production is low, and the production of microalgal biomass should ideally more or less match the gen­eration of wastewater. Such spatial and temporal mismatches between availability of wastewater and microalgal productivity may limit the potential to convert wastewater nutrients into microalgal biomass. Large cities generate enormous volumes of wastewater that could be used for microalgae biomass production. These cities, however, often lack sufficiently large areas of low-cost land nearby that can be used for microalgae production (Fortier and Sturm 2012). In addition, many of the world’s largest cities are situated in temperate climates where micro­algal productivity is low in winter and freezing temperatures may even require complete shutdown of microalgal farms for a few months (Chiu and Wu 2013). At high latitudes in winter, it may be impractical to use wastewater for microalgae production (Van Harmelen and Oonk 2006), and at low latitudes, high temperatures may also limit productivity during the warmest months.

Availability of land is probably less problematic when animal manure is used for microalgae production than when domestic wastewater is used. Livestock farms are generally situated away from cities. In general, agricultural land is available that can be converted in microalgae cultivation ponds. In many countries, however, there is an ongoing debate whether microalgae cultivation is allowed on agricultural land or not (Trentacoste et al. 2014). Due to economies of scale, we can assume that the minimum size of a microalgae farm would be about 10 ha (Lundquist et al. 2010). A 10-ha farm that produces 300 ton dry microalgal biomass ha-1 year-1 consumes 27 ton N year 1. This corresponds to the N output of a municipality of 9000 inhabitants, or a farm with at least 7000 pigs or 400 cattle. Wastewater from smaller farms or villages may be collected and transported to a microalgae farm, but this is only possible over relatively small distances due to the high cost for transporting wastewater. Therefore, smaller and isolated sources of wastewater will unlikely be practical for microalgae production.

TALENs comprise a powerful class of tools that are redefining the boundaries of biological research. TALEs are naturally occurring proteins from the plant patho­genic bacteria genus Xanthomonas, and contain DNA-binding domains composed of a series of 33-35 amino acid repeat domains that each recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids that are known as the repeat variable di-residues (RVDs). TALEs can be quickly engineered to bind practically any desired DNA sequence (Boch 2011). TALENs can be used to edit genomes by inducing double-strand breaks (DSB) (Fig. 8.2b), which cells respond to with repair mechanisms (Boch 2011). Several methods have been developed that enable rapid assembly of custom TALE arrays such as golden gate cloning, high-throughput solid-phase assembly, and ligation-independent cloning techniques (Gaj et al. 2013).

Site-specific nucleases have enabled the introduction of targeted modifications in several model organisms common to biological research, including zebrafish, rat, mouse, Drosophila, Caenorhabditis elegans, and many other non-model species including the monarch butterfly, frogs and livestock (Gaj et al. 2013). In addition to valuable animal models, TALENs have been used to introduce targeted alterations in plants, including Arabidopsis and several crop species (Curtin et al. 2012), allowing the incorporation of valuable traits, such as disease and herbicide resistance.

In algae, studies of TALENs to modify the genome of Chlamydomonas have been initiated (Borchers et al. 2012; Spalding and Wright 2011). For instance, TALENs to knockout the Chlamydomonas Sta6 and CAH3 genes, which are responsible for starch production and CO2 uptake, have been designed. These early studies are of interest due to the status of Chlamydomonas as a model organism for biofuel production.

The diversity of organisms modified by these site-specific nucleases will undoubtedly continue to grow, expanding the repertoire of model systems for basic research. TALENs will also enhance research efforts in algal biomass production, thus opening new avenues for algal biofuels commercialization.

Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (brand of Thermo Fisher Scientific, Carlsbad, CA, USA).

Apart from C. reinhardtii, few algal species have been subjected to extensive genomic manipulation. As it seems unlikely that C. reinhardtii will be used for commercial biofuel applications, this needs to be remedied. Because of the phy­logenetic and structural diversity of algae, methods established for C. reinhardtii cannot necessarily be easily transferred to other species and may require major adaptations. Therefore, recent efforts have been made to develop molecular toolkits to increase the range of other more suitable algal species for commercial production scenarios.

A number of algae species have been transformed successfully, and an overview is given in Table 11.2. For example, Euglena gracilis was transformed with an antibiotic resistance marker (Doetsch et al. 2001) and Porphyridium spp. using a herbicide resistance cassette (Lapidot et al. 2002) RNAi has also been used to engineer nuclear genes in the chlorophyte Dunaliella salina (Sun et al. 2008) and in the diatom Phaeodactylum tricornutum (De Riso et al. 2009). Applicable genetic modifications of green algae for industry are the transformation of Haematococcus pluvialis (Steinbrenner and Sandmann 2006; Teng et al. 2002), an important

(continued)

Methods A Agrobacterium, B biolistic bombardment, E electroporation, G glass bead agitation, S silicon carbide whiskers, PT protoplast transformation, and PEG with polyethylene glycol

producer of astaxanthin, and Dunaliella salina (Feng et al. 2009; Geng et al. 2004; Sun et al. 2005; Tan et al. 2005) used for P-carotene production. Diatoms are also important commercial sources for aquaculture feedstock, specialty oils such as omega-3 fatty acids, and are used in nanotechnology due to their unique silica frustules. There has been one report of a nuclear transformation of dinoflagellates (ten Lohuis and Miller 1998). Red algae have been used for both chloroplast transformation (Lapidot et al. 2002) and nuclear transformation (Cheney et al. 2001; Minoda et al. 2004). A human growth hormone (hGH) has been successfully expressed in the nucleus of Chlorella vulgaris (Hawkins and Nakamura 1999) and a fish growth hormone (GH) in Nannochloropsis oculata (Chen et al. 2008). Transformation techniques using a cellulolytic enzyme to weaken the cell walls and make the cells more competent for the uptake of foreign DNA have been suc­cessfully applied to the green algae Chlorella ellipsoidea (Liu et al. 2013) and may be applicable for the transformation of other algal species with tough cell walls in future. A synthetic biology approach to engineer complex photosynthetic traits from diverse algae into a more controllable production strains has been shown using an ex vivo genome assembly to transfer genes for core photosystem subunits from Scenedesmus into multiple loci in the Chlamydomonas plastid genome (O’Neill et al. 2012).

Given the recent expansion of interest in microalgae, a broader repertoire of genome sequences and analytical and molecular engineering tools are being reported and will provide the foundation for a broad range of biofuel applications, some of which are covered in the following section.

The use of an electric field to modify the surface charge of the algal cells and stimulate flocculation holds the potential for improved harvesting since, in theory, the method would not require addition of chemicals and could be easily run as a continuous system. The hurdles for general applicability of these technologies are development of electrodes that do not add metals to the system and reducing the cost of the power required.

Electrocoagulation using metal electrodes employs similar chemical phenomena as tradition coagulation, but rather than introducing a dry chemical, such as alu­minum chloride, metal ions are typically released from the reactive metal electrode into the water through electrolysis. The metal ions then act similarly to the dry chemicals. The use of electricity also helps influence the particle charges. Envi­ronmental parameters such as pH and salinity must be optimized for electrocoag­ulation to work efficiently. Therefore, the trade-offs between electrocoagulation and chemical methods are (1) higher energy cost but lower raw material cost assuming the same environmental adjustment, (2) fewer introduced counterions (e. g., chlo­rides), and (3) replacement costs for sacrificial plates.

One study used aluminum anodes for electrocoagulation of algae from a wastewater treatment plant. This study found that removal after 15 min reached as high as 99.5 % (based on chlorophyll content) with a power input of 550 W (Azarian et al. 2007). At a reduced energy usage (100 Wdm-3), the same separation efficiency could be achieved in 30 min. They did not analyze the Al ion released but recommended this as a further issue in need of optimization before the wide use of this method.

Electroflocculation using inert electrodes does not add metal ions to the culture since the electrodes are inert. The electric force is used to drive cells to the anode where the cells lose charge and flocculate. One study showed that 80-95 % of the algal cells from a wastewater treatment plant could be removed from a 100-L vessel in 35 min (Poelman et al. 1997). These techniques are typically higher in both operating and capital costs due to higher energy requirement and more valuable metals.

Bailliez et al. (1986) found that the immobilized B. braunii cultures in calcium alginate beads had higher chlorophyll and photosynthetic activities compared to their free cells. S. obliquus cells immobilized in alginate (Brouers et al. 1983), and C. vulgaris and Anacystis nidulans in agar (Kayano et al. 1981; Weetall and Krampitz 1980), also showed a significant increase in their chlorophyll content. Enhanced chlorophyll and photosynthetic activity was explained by the protection of immobilized cells from photoinhibition due to the self-shadowing effect, and a possible increase in the concentrations of particular ions in the microenvironment of cells which can improve photosynthesis (Bailliez et al. 1986; Tamponnet et al. 1985).

Individually co-immobilized cells of C. vulgaris and C. sorokiniana with A. brasilense growth-promoting bacterium also yielded higher chlorophyll a and b, violaxanthin, and lutein accumulation compared to the immobilized algal cells without any bacterium (de-Bashan et al. 2002a).

Lebeau et al. (2000) reported that the immobilization of the marine diatom Haslea ostrearia in agar had a positive effect on the continuous production of the marennin pigment, which is primarily used for the oyster-breeding industry.

Some potential limitations of the secondary product formation by immobilized cells are the commonly reported slower growth rates of the microorganisms compared to their free-cell suspension systems and slower diffusion rates of the target-products (i. e., hydrogen) from the cells into their environment. Resolving these issues with the combination of optimized immobilization matrices and innovative bioreactor designs (e. g., some attempts include membrane-based cell recycle bioreactor (Chang et al. 1994); dual-layer coaxial hollow fiber-type biore­actor (Yang et al. 2006); and a multimembrane bioreactor in a pressure cycling mode (Efthymiou and Shuler 1987), aiming to increase the nutrient transfer to the cells) can potentially bring other dimensions to the research areas of those afore­mentioned bioprocesses.

Two criteria that drive strategies for algae-based wastewater treatment for biofuel production are the need to produce a high-quality effluent on a consistent basis and the need to produce biomass with high oil content. While there are proponents for hydrothermal liquefaction (HTL) treatment of algal biomass to develop a “green crude,” the goal in the scenario described above is to separate the oil and the biomass. The nitrogen and phosphorus content captured in the biomass is needed in the production of methane in the digester, and later, the biosolids from the digester can be composted with green waste to make a high-quality fertilizer.

To meet both criteria, compromises must be made in the selection of algae. In traditional wastewater oxidation ponds, there are a wide array of different pro­karyotic and eukaryotic photosynthetic organisms. These ponds are subject to seasonal shifts in dominant populations as well as changes due to predation by protozoans and zooplankton. The variation in algal population dynamics can be minimized by periodic inoculation of the pond with a desired unialgal strain cul­tured in photobioreactors. Another consideration is the relationship between bac­teria in the wastewater and the algae. In addition, evidence exists for a role for heterotrophic microbes in algae auto-flocculation (Lundquist et al. 2011). A close examination of algae collected from wastewater ponds using a light microscope will usually show a significant number of heterotrophs associated or attached to the surface of the algae. If the algae are to be co-cultivated in photobioreactors as described above, it would be prudent to include the strains of wastewater bacteria associated with the specific type(s) of algae and which promote flocculation. The microalgae encompass a phylogenetically diverse assemblage of prokaryotic and eukaryotic photosynthetic microorganisms found in a wide range of habitats ranging from terrestrial environs to fresh and marine to hypersaline waters. It follows then that the ecology, morphology, biochemistry, and physiology are also diverse. Although the number of algal species is estimated to range between 30,000 and 300,000 with 7500 species systematically estimated from the literature (Guiry

2012) , less than 1 % have been isolated and characterized (Radner and Parker

1994) . Thus, the biotechnological potential of these microorganisms is just beginning to be explored for production of high-value and value-added products and biofuels. Microalgae have been used for decades as a source of high-value compounds with pharmaceutical activity including anticancer, antimicrobial, anti­viral agents, and pigments including a variety of carotenoids, cosmetics, nutra — ceuticals, and feed supplements for poultry, livestock, and mariculture (see Walker et al. 2005 for a review). Many groups are now exploring the use of transformable eukaryotic strains to produce heterologous proteins since they are capable of intron- splicing, glycosylation, and multimeric protein assembly (Spolaore et al. 2006).

Several aspects of algae biology and physiology are relevant to their economic potential of microalgae as a feedstock for biodiesel coupled to bioremediation. Of particular interest to the biofuels industry are productivity, biochemical composi­tion, and the influence of environmental and cultural practices on physiological processes, especially lipid metabolism and its regulation, photosynthetic efficiency, cell wall structure, and heterotrophic/mixotrophic capabilities. In addition, tech­niques to control algal/microbial pond community structure are essential for quality control of biodiesel composition from algae biomass. The ASTM International consensus-based standards group, whose standards are recognized in the United States, have specifications for the quality of biodiesel. The fuel characteristics are strongly influenced by FAME composition including chain length and degree of saturation. FAMEs isolated from algae species range in size from 12 to 38 carbons. The hydrocarbons that comprise petroleum products range in length as follows: 5-12 carbons for gasoline, 10-15 for diesel fuel, and 12-16 for kerosene (the main component of jet fuel). Refineries crack the longer hydrocarbons found in crude oil, then distill and blend the resulting compounds to formulate standard petroleum products. Maintenance of species composition, especially in outdoor ponds and when using wastewaters, is problematic. Cultivation of Spirulina in open outdoor ponds has been a success story in commercial algaculture. This strain grows in nearly pure culture in the alkaline, high-salinity waters of Lago de Texcoco. Competition from invading species is minimized due to the inhospitable nature of these waters. Control of species composition is crucial to quality control of biodiesel production. Lipid profiles are characteristic of some organisms and have even been used as a taxonomic feature. However, microalgae show great inter — and intraspecific variation in fatty acid profiles and these profiles can be affected by culture conditions (Roessler 1990).