Production and Harvesting of Microalgae for Wastewater Treatment, Biofuels, and Bioproducts

Biotechnology Advances 29 (2011) 686–702

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Biotechnology Advances

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Research review paper Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts

Logan Christenson, Ronald Sims ⁎

Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, United States article info abstract

Article history: The integration of microalgae-based biofuel and bioproducts production with wastewater treatment has Received 18 March 2011 major advantages for both industries. However, major challenges to the implementation of an integrated Received in revised form 21 May 2011 system include the large-scale production of algae and the harvesting of microalgae in a way that allows for Accepted 27 May 2011 downstream processing to produce biofuels and other bioproducts of value. Although the majority of algal Available online 2 June 2011 production systems use suspended cultures in either open ponds or closed reactors, the use of attached cultures may offer several advantages. With regard to harvesting methods, better understanding and control Keywords: fl fl Algae production of auto occulation and bio occulation could improve performance and reduce chemical addition Algae harvesting requirements for conventional mechanical methods that include centrifugation, tangential filtration, gravity Biofuel sedimentation, and dissolved air flotation. There are many approaches currently used by companies and Wastewater treatment industries using clean water at laboratory, bench, and pilot scale; however, large-scale systems for controlled Photobioreactor algae production and/or harvesting for wastewater treatment and subsequent processing for bioproducts are Raceway lacking. Further investigation and development of large-scale production and harvesting methods for biofuels fi Algae bio lm and bioproducts are necessary, particularly with less studied but promising approaches such as those involving attached algal biofilm cultures. © 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 687 2. Major challenges ...... 687 2.1. Nutrient supply and recycling ...... 688 2.2. Gas transfer and exchange ...... 689 2.3. PAR delivery ...... 689 2.4. Culture integrity ...... 689 2.5. Environment control ...... 689 2.6. Land and water availability ...... 689 2.7. Harvesting ...... 690 2.8. Genetic and metabolic engineering ...... 690 2.9. Summary of major challenges ...... 690 3. Algae production methods ...... 690 3.1. Suspended cultures ...... 690 3.1.1. Open ponds ...... 690 3.1.2. Closed reactors ...... 691 3.2. Immobilized cultures ...... 691 3.2.1. Matrix-immobilized microalgae ...... 691 3.2.2. Algal bioﬁlms...... 691 3.3. Algae production cost ...... 692 4. Algae harvesting methods ...... 692 4.1. Chemical based ...... 692 4.2. Mechanical based ...... 693

⁎ Corresponding author. Tel.: +1 435 797 2785; fax: +1 435 797 1248. E-mail address: [email protected] (R. Sims).

4.3. Electrical based ...... 693 4.4. Biological based ...... 693 5. Approaches to algae production and harvesting in industry ...... 694 5.1. Reactor designs for algae production ...... 694 5.1.1. Open ponds ...... 694 5.1.2. Closed reactors ...... 694 5.1.3. Hybrid designs ...... 697 5.1.4. Bioﬁlm reactors ...... 697 5.2. Harvester designs and harvesting processes ...... 697 5.2.1. Mechanical harvesters ...... 697 5.2.2. Biological based harvesting ...... 697 5.2.3. Bypassing the harvesting step ...... 698 5.3. Process design ...... 698 5.3.1. Culture control ...... 698 5.3.2. Lipid accumulation ...... 698 5.3.3. Nutrient and gas recycle ...... 698 5.4. Genetic manipulation ...... 698 5.5. Summary of approaches in industry ...... 699 6. Conclusions ...... 699 References ...... 700

1. Introduction without sufficient production and harvesting of the algae crop. Unfortunately, no current approach has been demonstrated to be simple With growing concerns surrounding the continued use of fossil fuels, and inexpensive enough for economical large-scale use with algae. renewable biofuels have received a large amount of recent attention. The U.S. Department of Energy has recognized the potential While biofuels produced using oil crops and waste oils cannot alone meet synergy of wastewater treatment and biofuel production from algae, the existing demand for fuel, microalgae appear to be a more promising stating that “Inevitably, wastewater treatment and recycling must be feedstock option (Chisti, 2007, 2008). Microalgae include microscopic incorporated with algae biofuel production (U.S. DOE, 2010).” Because eukaryoticalgaeaswellascyanobacteria(Acreman, 1994). Such algae much of the infrastructure is already in place, algae based wastewater could provide substantially more biodiesel than existing oilseed crops treatment can be deployed relatively soon. The use of wastewater can while using less water and land (Sheehan et al., 1998). Algae biomass offset the cost of commercial fertilizers otherwise needed for the may also be fed to an anaerobic digester for methane production production of algae, and wastewater treatment revenues can offset (Golueke et al., 1957; Gunaseelan, 1997; Yen and Brune, 2007), or used to algae production costs. It is apparent that overcoming the current produce bioplastic materials (Chiellini et al., 2008). Residual biomass challenges to the production and harvesting of algae will be beneficial from these processes can potentially be used as a fertilizer, soil for both wastewater treatment and for the production of biofuels and amendment, or feed for fish or livestock (Mulbry and Wilkie, 2001; bioproducts. Mulbry et al., 2005; Roeselers et al., 2008). However, the production of Considering the benefits of cost-effective algae production and biofuels and bioproducts using algal biomass has been handicapped by an harvesting to both wastewater treatment and the production of inability to find a reliable and cost effective method of producing and biofuels and other bioproducts, this review has the following harvesting large quantities of algae feedstock. objectives: In addition to biofuel and other bioproduct applications, large- scale methods of producing and harvesting algae have uses in 1. Identify the major challenges to cost-effective production and wastewater treatment (Hoffmann, 1998; Oswald, 2003). Without harvesting of algae. proper treatment, excess nitrogen and phosphorus in discharged 2. Compare the benefits and limitations of the different approaches to wastewaters can lead to downstream eutrophication and ecosystem algae production, including open ponds, closed reactors, and damage (Correll, 1998). The negative effects of such nutrient over- immobilized systems. loading of receiver systems include nuisance algae, low dissolved 3. Compare the benefits and limitations of algae harvesting ap- oxygen concentrations and fish kills, undesirable pH shifts, and proaches, including chemical, mechanical, biological, and electrical cyanotoxin production. While chemical and physical based technol- based harvesting. ogies are available to remove these nutrients, they consume 4. Examine algae production and harvesting approaches in industry. significant amounts of energy and chemicals, making them costly 5. Identify research needs and potential solutions to the major processes (Tchobanoglous and Burton, 1991). Chemical treatment challenges of production and harvesting of algae. often leads to secondary contamination of the sludge byproduct as well, creating additional problems of safe disposal (Hoffmann, 1998). The energy and cost required for tertiary treatment of wastewater 2. Major challenges remain a problem for industries and municipalities. Compared to physical and chemical treatment processes, algae based The two major challenges to the implementation of an integrated treatment can potentially achieve nutrient removal in a less expensive algae system include the large-scale production of algae and the and ecologically safer way with the added benefits of resource recovery harvesting of algae in a way that allows for downstream processing to and recycling (LE Graham et al., 2009; Oswald, 2003). Common nitrogen produce biofuels and other bioproducts of value. The challenges with removal methods such as bacterial nitrification/denitrification remove regard to large-scale production of algae include nutrient supply and the majority of the nitrogen as N2 gas, whereas algal treatment retains recycling, gas transfer and exchange, photosynthetically active useful nitrogen compounds in the biomass. Notwithstanding these radiation (PAR) delivery, culture integrity, environment control, benefits, acceptable nutrient levels in the effluent cannot be achieved land and water availability, and harvesting. 688 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702

Algae growth requires the availability of primary nutrients and (Table 1) for a total of 77.6 million kg day−1. Assuming 90% removal of micronutrients, which can be costly if they need to be added in great the limiting nutrient, a 10% (w/w) biodiesel yield [see lipid content of amounts. When gas exchange is insufficient, the algae culture can mixed cultures grown in municipal wastewater as reported by Woertz become carbon limited, and the oxygen byproduct of photosynthesis et al., 2009], a biodiesel density of 0.801 kg l−1 (Vijayaraghavan and can reach inhibitory levels (Carvalho et al., 2006). Delivery of light in Hemanathan, 2009), and 9 months per year operation, an average the form of photosynthetically active radiation (PAR) can also be the biodiesel production of roughly 1.7 million gallons day−1 is calculated limiting factor at high culture densities (Tredici and Zittelli, 1998; (6.5 million liters day−1). Although this is only a small fraction of the Zijffers et al., 2008). Depending on the characteristics of the 378 million gallons of transportation fuel the U.S. uses per day (U.S. EIA, microalgae culture, contamination can be difficult to avoid. Increasing 2009), wastewater treatment plants still appear to be the best training control of the growth environment can enhance productivity but grounds for the development of any commercial scale algae biofuels involves additional costs. Sufficient land and water must also be facilities in the future. available. The most pressing challenge, however, lies not in the Using algae to simultaneously treat wastewater and produce production of the algae crop, but in the harvesting and downstream biofuels is also justified by the potential to offset the high costs of processing of it in a manner suitable for the production of bioproducts wastewater treatment. The U.S. has wastewater infrastructure (Molina Grima et al., 2003; Uduman et al., 2010). Each of the investment requirements of $13 billion to $21 billion annually with challenges identified above is addressed in the following subsections. an additional $21.4 billion to $25.2 billion required for annual operation and maintenance (U.S. CBO, 2002). In addition to revenues 2.1. Nutrient supply and recycling from the biodiesel produced by wastewater grown algae, anaerobic digestion of the leftover biomass can produce biomethane for Growing algae requires consideration of three primary nutrients: electricity generation. Using the same biomass production calcula- carbon, nitrogen, and phosphorus. Micronutrients required in trace tions as above, and assuming a methane yield of 0.3 l g− 1 for biomass amounts include silica, calcium, magnesium, potassium, iron, man- after oil extraction (Lundquist et al., 2010), a methane heating value of ganese, sulfur, zinc, copper, and cobalt, although the supply of these 55,500 kJ kg− 1 (NIST, 2011), and an electricity generation efficiency essential micronutrients rarely limits algal growth when wastewater of 30%, 70.6 GWh day− 1 of cost offsetting electricity can be produced is used (Knud-Hansen et al., 1998). If not already available in the from algae grown in U.S. wastewater treatment plants. water source, the addition of commercial fertilizers can significantly Although available carbon can be the limiting factor, the increase production costs, making the price of algae derived fuel cost atmosphere provides a near infinite, although slowly transferred, prohibitive (U.S. DOE, 2010). For this reason, wastewater is an source of carbon dioxide. Nitrogen and phosphorus, therefore, are the attractive resource for algae production. two nutrients of most concern when analyzing a water source for Pittman et al. (2011) reviewed the potential of algal biofuel potential algae growth. To prevent limitations by either, the molar production and concluded that, based on current technologies, algae ratio of the water supply must match the stoichiometric ratio of the cultivation for biofuels without the use of wastewater is unlikely to be algae biomass. This nitrogen to phosphorus ratio is often assumed to economically viable or provide a positive energy return. Lundquist et al. match the Redfield ratio of 16:1 (Stumm and Morgan, 1981). This (2010) analyzed several different scenarios of algae-based wastewater ratio is not a universal biochemical optimum, but instead represents treatment coupled with biofuel production and concluded that only an average of species specific N to P ratios that vary from 8 to 45 those cases that emphasized wastewater treatment were able to (Klausmeier et al., 2004). This means that even when wastewater is produce cost competitive biofuels. They concluded that the near-term used to supply nutrients, addition of nitrogen or phosphorus may be outcome for large scale algae biofuels production is not favorable needed in order to reach the proper ratio. without wastewater treatment as the primary goal. Nutrient starvation can be intentionally designed into a process as a According to the 2008 Clean Watersheds Needs Survey, the total of method of increasing the value of the algae biomass. Much of the focus reported wastewater flow in the U.S. is 32,345 million gallons per day of the Department of Energy's Aquatic Species Program was on (MGD) (U.S. EPA, 2008). Using medium strength domestic wastewater enhancing lipid production within the cells through stress conditions values, there is enough N and P in each liter to produce 0.6 g of algae such as nitrogen deficiency. This often led to higher lipid accumulation,

Table 1 Characterization of typical wastewaters with respect to algal nutrients nitrogen and phosphorus.

Wastewater type Nitrogena (mg l− 1) Phosphorusb (mg l− 1) Reference N:P (molar ratio) Theoretical algae biomass productionc,d

Weak domestic 20e 4 Tchobanoglous and Burton (1991) 11 0.3 g Medium domestic 40e 8 Tchobanoglous and Burton (1991) 11 0.6 g Strong domestic 85e 15 Tchobanoglous and Burton (1991) 13 1.4 g Beef cattle feedlot 63 14 Bradford et al. (2008) 10 1.0 g Dairy 185 30 Bradford et al. (2008) 14 2.9 g Poultry feedlot 802 50 Bradford et al. (2008) 36 5.7 g Swine feedlot 2430 324 Bradford et al. (2008) 17 37.1 g Swine feedlot 895 168 Vanotti and Szogi (2008) 12 14.2 g Coffee production 85 38f Olguin et al. (2003) 5 1.3 g Coke plant 757 0.5f Vazquez et al. (2007) 3352 0.1 g Distillery 2700e 680f Basu (1975) 9 42.8 g Paper mill 11e 0.6 Pokhrel and Viraraghavan (2004) 41 0.1 g Tannery 273 21f Durai and Rajasimman (2011) 29 2.4 g Textile 90 18 Fongsatitkul et al. (2004) 11 1.4 g Winery 110 52 Mosse et al. (2011) 5 1.7 g

a Total Kjeldahl nitrogen (TKN) unless specified. b Total phosphorus unless specified. c Based on limiting nutrient assuming a formula of C106H181O45N16P(Stumm and Morgan, 1981). d Based on the nutrients (N and P) contained in one liter of wastewater. e Total nitrogen. f Phosphorus as phosphate (PO4–P). L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 689 but these gains were more than offset by the slower growth rates and 2.3. PAR delivery did not lead to an overall increase in lipid production (Sheehan et al., 1998). PAR is different from other algae growth requirements in that it Table 1 shows typical N and P characteristics of several different cannot be mixed. Full sunlight is often too intense for algae to utilize wastewater types, and the theoretical algae production from the all available photons, and excess energy absorbed by cells is lost in the nutrients available in these waters. Domestic wastewater treatment form of fluorescence or heat (Niyogi, 2003; Powles, 1984). Not only is plants, confined animal feeding operations (CAFOs), and the other this inefficient use of available light, but prolonged exposure to such listed industries of Table 1 are good candidates for algae-based high intensities can overpower the energy dissipating machinery of treatment due to the respective wastewater compositions and the the cells resulting in photoinhibition and cell damage (Niyogi, 2003; existing need to treat these waste streams. Although some of these Powles, 1984). In contrast, algae in deeper portions of a culture are wastewaters typically contain organics and/or heavy metals, algae- often light limited because the majority of light has already been based treatment may also aid in the removal of these constituents. absorbed by the outermost layer of cells (Borowitzka, 1999). Thus, Heavy metals can be removed by adsorption to algae cells, and algae cultures often suffer from photoinhibition and photodeprivation can be responsible for the degradation of organics such as phenol and simultaneously. acetonitrile (U.S. DOE, 2010). Increasing the utilization of PAR is usually dealt with by designing the reactor with high surface area to volume ratio and/or vigorous mixing to ensure that all cells reside in the illuminated area for an 2.2. Gas transfer and exchange appropriate length of time. Hu et al. (1996) and Hu and Richmond (1996) have shown high culture densities using well mixed flat panel Proper gas exchange for algae growth includes both sufficient reactors with high surface area to volume ratio. Degen et al. (2001) transfer of carbon dioxide to the cells and sufficient removal of oxygen were able to show 1.7 times greater productivity simply by placing gas. Although some algae can be grown heterotrophically, an baffles in an air lift reactor to better manage the light/dark frequency environmentally and economically viable process must make use of of the culture. algae's autotrophic abilities by using inorganic carbon as the carbon source. The three principle forms of dissolved inorganic carbon (DIC) 2.4. Culture integrity associated with algal growth exist in equilibrium as carbon dioxide, bicarbonate, and carbonate. Algae can directly utilize carbon dioxide In monocultures grown for nutritional supplements or other and often bicarbonate, but generally not carbonate (Knud-Hansen et al., bioproducts, algal cultures are susceptible to contamination by less 1998; Round, 1984). desirable strains unless additional means of control are utilized (U.S. Open ponds can potentially be carbon limited due to mass transfer DOE, 2010). Monocultures of high lipid producing strains are likely to fi limitations. Azov (1982) recommended arti cially maintaining high be outcompeted by faster growing species of microalgae or cyano- free carbon dioxide concentrations in outdoor algae cultures after bacteria (Vasudevan and Briggs, 2008). Carefully maintained mono- fi – nding that cultures at higher levels had 65 95% more dry weight cultures are not found in wastewater treatment systems. When than the control. Increases in lipid content have also been shown with wastewater resources are used, naturally occurring mixed cultures of fi carbon dioxide addition (Chiu et al., 2009; Grif ths, 2009). Simply algae dominate. Although culture composition and growth conditions bubbling carbon dioxide into the culture, however, may not be may be less manageable, lipid accumulation of mixed cultures in effective enough, as bubble residence time may be too short, and municipal wastewater has been shown to reach 11.3% (Woertz et al., much can end up being lost to the atmosphere (Mata et al., 2010). 2009), and as high as 29% when grown with anaerobic digester Optimizing carbon dioxide delivery through direct bubbling or other effluent (Woertz et al., 2009). Griffiths (2009) reported a fatty acid means remains an engineering challenge. In addition, good sources of methyl ester content of as high as 23.4% after in-situ transesterifica- fl highly concentrated carbon dioxide, such as from ue gas, are not tion of a mixed culture grown in municipal wastewater. always near enough to wastewater sources to justify the cost of transfer and use. According to Lundquist et al. (2010), well below 10% 2.5. Environment control of the CO2 resource base is available for algae growth due to climatic, water, and land limitations. Efficiently bringing together waste carbon dioxide and waste nutrients is part of the challenge to cost-effective Both biomass production and nutrient removal can be optimized if production of algae. the important growth parameters such as temperature and pH are A challenge directly related to carbon dioxide supply is the better controlled (Abu-Rezq et al., 1999). More control over the removal of excess oxygen. Oxygen concentrations above air saturation growth environment includes additional costs, however, such as with begin to inhibit photosynthesis, and this byproduct must be removed the use of closed reactors instead of open ponds (Shen et al., 2009). in order to prevent photooxidative damage. For closed reactors Concerning wastewater treatment ponds and lagoons, the large scales especially, oxygen removal is considered one of the most difficult involved lessen available means of environmental control. Finding challenges to overcome (Carvalho et al., 2006). ways to achieve proper control of the growth environment without Even when atmospheric carbon dioxide is the only available adding unreasonable costs remains a challenge. source, methods can be employed to increase transfer to the liquid phase. Both carbon dioxide transfer and oxygen release can be 2.6. Land and water availability increased through the use of gas–liquid contactor reactors such as rotating biological contactors (RBCs) common in secondary waste- Large scale production of microalgae likely requires a large water treatment (Zeevalkink et al., 1979). Patwardhan (2003) expanse of land with an available water source. Wastewater reported that RBC systems show much higher gas transfer efficiency treatment facilities have plenty of nutrient rich water available, but than surface aerators, diffuser aerators, or trickling filters. Putt (2007) may not have the necessary land, especially considering newer showed that a wetted ramp contactor would increase the carbon membrane reactor facilities designed to leave a small footprint. uptake of a pond by a factor of 36 relative to a regular pond, although Regardless, Sheehan et al. (1998) concluded that at least in the United he concluded that this was still not sufficient enough. Cost effectively States, land is definitely not a limitation, and although the technology delivering carbon dioxide while allowing adequate oxygen release faces many research and development hurdles, resource limitation is remains a challenge. not a valid argument against further development. 690 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702

According to the United States Environmental Protection Agency, production (Sheehan et al., 1998). By producing strains deficient in there are more than 7000 facultative lagoon systems in the United ADP-glucose pyrophosphorylase, a few studies have shown increased States (U.S. EPA, 2002). From the perspective of algae production, lipid content in Chlamydomonas reinhardtii by reducing the amount of lagoon treatment facilities provide the combined benefits of land, carbon channeled to starch production (Li et al., 2010; Wang et al., water, and nutrient availability, with reduced need for preliminary 2009). site construction and infrastructure development. For these reasons, Mussgnug et al. (2007) have also produced a modified strain of C. lagoons stand out as promising potential algae production facilities. reinhardtii. By downregulating the expression of light harvesting One such facility is the Logan Regional Wastewater Treatment Plant, complex proteins, a 30% improvement in photosynthetic efficiency located in northern Utah. The plant consists of 460 acres of lagoons, was achieved. By reducing the amount of light wasted as heat and and facility directors are dedicated to an algae-based approach to fluorescence, such an improvement has ramifications for reactor wastewater treatment with additional production of bioproducts design, potentially increasing the allowable culture depth and (Griffiths, 2009). reducing mixing requirements. Seawater or saline aquifer water has also low value water sources Other strategies being investigated include the downregulation of that can be used for algae production (Hu et al., 2008; Schenk et al., enzymes involved in lipid catabolism, expression of certain acyl-ACP 2008). Compared to wastewater, the major disadvantage of these thioesterases for a higher proportion of fatty acids with diesel quality water sources is the lower N and P content. Fertilizer addition chain length, and direct synthesis of fuels, ideally coupled with accounts for 6–8 cents per gallon additional operating cost (in 1987 U. secretion of the fuel product by the genetically modified algae S. dollars) assuming 50% nutrient recycle (U.S. DOE, 2010). An algae (Radakovits et al., 2010). See also Section 5.4 for a discussion of production operation not based on wastewater will also lose the genetic manipulation in the algae production industry. benefit of wastewater treatment credits. Lundquist et al. (2010) predicted a 10% increase in operation cost if non-wastewater sources 2.9. Summary of major challenges are used, but an overall increase of 20–25% when lost wastewater credits were considered. However, as discussed in Section 2.1, the use Several challenges remain in the development of a large-scale of wastewater resources alone will not be enough to satisfy the algae production and harvesting system. The use of existing demand for fuel, and other low value water sources will need to be wastewater lagoons can resolve many of the challenges discussed, utilized for algae biofuels to expand beyond the initial wastewater including nutrient supply and recycling as well as land and water niche. availability, but of the thousands of existing lagoons, few harvest algae (Salerno et al., 2009), and those that do favor processes involving 2.7. Harvesting chemical coagulants (Friedman et al., 1977; Hoffmann, 1998; Teixeira and Rosa, 2006). Other than preliminary research at Utah State Separating the algae from water remains a major hurdle to University (Griffiths, 2009) and California Polytechnic State Univer- industrial scale processing partly because of the small size of the algal sity (Woertz et al., 2009), little has been done to produce biofuels and cells, with unicellular eukaryotic algae typically 3–30 μm(Molina bioproducts from algae grown in wastewater. Grima et al., 2003), and cyanobacteria as small as 0.2–2 μm(Chorus and Bartram, 1999). In addition, relatively dilute cultures of 200– 3. Algae production methods 600 mg/l are common (Uduman et al., 2010), and require that large volumes of water be processed. Recovery has been estimated to Suspended cultures, including open ponds and closed reactors, and contribute 20–30% of the total cost of producing the biomass (Molina immobilized cultures, including matrix-immobilized systems and Grima et al., 2003). The initial harvesting step is not only costly, but biofilms, are addressed in the following sections. Table 2 compares also affects any later processes downstream. open ponds, closed reactors, and biofilm systems against scalability Most wastewater treatment lagoons in the U.S. do not harvest and operating parameters. algae (Salerno et al., 2009). Middlebrooks et al. (1974) reviewed several removal methods suitable for wastewater lagoons and 3.1. Suspended cultures recommended granular media filters for communities with smaller ponds. At plants that do remove algae, chemical coagulation followed The greatest amount of information on how to treat wastewater by sedimentation or dissolved air flotation (DAF) is a common with algae pertains to suspended algae systems comprised of approach (Friedman et al., 1977), with DAF generally considered more naturally occurring mixed cultures. Most methods of producing effective than sedimentation in the treatment of algae rich waters algae for the purpose of biofuels are also based on suspended algae. (Teixeira and Rosa, 2006). Though effective at full scale, the addition Table 3 shows biomass productivity and wastewater nutrient removal of chemical coagulants transforms a potential resource into waste by suspended culture designs. sludge that must be disposed of (Hoffmann, 1998). Lowering the cost of harvesting algae and harvesting in a way that allows for the 3.1.1. Open ponds creation of bioproducts remains a challenge. The most common large scale production systems in practice are high rate algal ponds, also known as HRAPs or raceway ponds. In use 2.8. Genetic and metabolic engineering since the 1950s, raceway ponds are open, shallow ponds with a

Many researchers are seeking to overcome the production and harvesting challenges through genetic and metabolic engineering of Table 2 microalgae. Using nutrient deprivation or other stresses to induce a Benefits and limitations of design approaches for algae production. Culture density fi natural lipid trigger is not always beneficial because productivity and gures adapted from Shen et al. (2009), Johnson and Wen (2010), U.S. DOE (2010), and Norsker et al. (2011). lipid accumulation are often inversely related. According to Hu et al. (2008), an increased understanding of the control mechanisms Design Culture density Gas exchange Scalability Culture control − 1 behind lipid production is needed to enable genetic manipulation (g l ) for simultaneous rapid growth and high lipid content. Acetyl-CoA Raceway pond 0.25–1 Low High Low carboxylase is the rate-limiting enzyme in fatty acid synthesis; Tubular reactor 1.5–1.7 Very low Medium High fi however, over-expression of this enzyme has not led to higher lipid Bio lm system 70 High High Low L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 691

Table 3 Algae biomass production and wastewater nutrient removal in suspended systems.

Design Nutrient loadinga Nutrient removal Biomass production Scale Reference (mg l− 1 day− 1) (g m− 2 day− 1)

Raceway pond P: 1.2–7.5 P: 96% 10–20 Pilot and demonstration Hoffmann (1998), Shen et al. (2009), Lundquist et al. (2010) Tubular reactor N: 17.3 N: 99% 20–45 Pilot and demonstration Chisti (2007), González et al. (2008), Shen et al. (2009) P: 1.4 P: 86%

a Soluble/dissolved forms of N and P. paddle wheel to provide circulation of the algae and nutrients. et al., 1985), as well as increased cellular pigment, lipid content, and Raceways are relatively inexpensive to build and operate, but often lipid variety (de-Bashan et al., 2002). For these immobilization suffer low productivity due to contamination, poor mixing, dark processes, however, such benefits are likely offset by the high cost zones, and inefficient use of CO2 (Chisti, 2007; Mata et al., 2010). of the immobilization matrix. Such designs have thus far been Raceway ponds should theoretically have production levels of 50– confined to the laboratory. At the scale necessary for wastewater 60 g m− 2 day− 1, and single day productivities at this level have been treatment and biofuel production, the cost of the polymeric matrix reported (Sheehan et al., 1998), but in practice, productivities of even becomes prohibitive (Hoffmann, 1998). 10–20 g m− 2 day− 1 are difficult to achieve (Shen et al., 2009). The high evaporation rate of open ponds is most often seen as a limitation, 3.2.2. Algal biofilms but it also helps somewhat with temperature regulation through Algal biofilms could play a large role in overcoming the major evaporative cooling (U.S. DOE, 2010). A major conclusion of cost challenges to production and harvesting of microalgae. The waste- analysis studies conducted by the U.S. Department of Energy's Aquatic water treatment industry is already accustomed to large scale biofilm Species Program was that there is little prospect for alternatives to the processes (Wuertz et al., 2003), and according to Middlebrooks et al. open pond system given the requirements for low cost of fuel (1974), if enough surface area is provided, algae biofilm growth can be (Sheehan et al., 1998). more than suspended growth. A scalable algal biofilm system could be integrated into the treatment process, thereby achieving the dual 3.1.2. Closed reactors benefits of inexpensive nutrient supply and treated water. Surface Tubular photobioreactors are the only type of closed systems used at attached algal biofilms can offer the same increased culture density large scale (Chisti, 2007). Vertical, horizontal, and helical designs are and lower land and water requirements of matrix-immobilized common, although helical designs are considered the easiest to scale up cultures (U.S. DOE, 1985) without the associated costs of the matrix. (Carvalho et al., 2006). Compared to open ponds, tubular photobior- Compared to suspended cultures, an algal biofilm system can better eactors can give better pH and temperature control, better protection integrate production, harvesting, and dewatering operations, poten- against culture contamination, better mixing, less evaporative loss, and tially leading to a more streamlined process with reduced down- higher cell densities (Mata et al., 2010). Reported productivities stream processing costs. generally range from 20 to 40 g m−2 day−1 (Shen et al., 2009). Despite Biofilm formation occurs due to the concentration of cations, these benefits, tubular reactors have not achieved significant use due to proteins, and organic molecules on submerged surfaces relative to the problems with toxic accumulation of oxygen, adverse pH and CO2 bulk aqueous environment, creating a favorable location for microbial gradients, overheating, bio-fouling, and high material and maintenance growth. Microbes colonizing a surface then secrete extracellular costs (Mata et al., 2010; Molina Grima et al., 1999). Oxygen removal is polymeric substance (EPS) composed of polysaccharides, proteins, considered one of the most difficult problems to overcome, especially nucleic acids, and phospholipids (Qureshi et al., 2005). when considering scale up, as it effectively limits tube or panel length Algae biofilms are likely to be benefited by bacteria present in and forces a more complex or modular design (Carvalho et al., 2006). wastewater. Hodoki (2005) showed that attached algae increased significantly when more bacteria were present on all substrata tested, 3.2. Immobilized cultures and Holmes (1986) saw that attachment of unialgal cultures with bacterial contaminants was one to two orders of magnitude higher Regardless of the specific advantages and disadvantages of than without bacteria. Both investigators theorized that entrapment raceways and tubular photobioreactors, both involve significant by attached bacteria is the major cause of early algal migration. challenges of biomass recovery. Because of the harvesting challenges Much of the research on algae biofilms has been associated with associated with suspended algae, there is a growing interest in the use limnological studies involving periphyton monitoring, often utilizing of immobilized or attached algal processes (Hoffmann, 1998). The U.S. artificial streams lined with Styrofoam (Bothwell, 1983; Sperling Department of Energy reviewed immobilized algae designs, mostly and Grunewald, 1969). In the wastewater treatment field, bacterial focusing on the use of immobilization particles in a packed or fluidized biofilm based reactors including trickling filters and rotating bed reactor (U.S. DOE, 1985). Although they reported that the biological contactors have been used successfully at large scales economics of such a scheme were prohibitive, they also concluded (Wuertz et al., 2003). Some research has been done to optimize that the benefits of increased culture densities and lower water and algae growth with these designs or incorporate them into an algae land requirements of immobilized algae systems could be realized growth process. Integrating a trickling filter after a raceway was through future design innovation (U.S. DOE, 1985). shown to aid in algae harvesting after the algae became entrapped in the biofilm of the filter (Hoffmann, 1998). Torpey et al. (1971) 3.2.1. Matrix-immobilized microalgae used artificially illuminated rotating aluminum disks to grow algae Results from experiments with algae immobilized in carrageenan for removal of nitrogen and phosphorus, and Przytocka-Jusiak et al. or alginate matrices have shown some potential benefits of (1984) used rotating Styrofoam disks to grow algae for ammonia immobilization, including efficient nutrient removal in wastewater removal; however, neither study attempted to harvest the algae or applications (Chevalier et al., 2000). According to Hameed and maximize production. Ebrahim (2007), results comparing growth rates of immobilized Cao et al. (2009) envisioned a floating conveyer belt system of cultures and suspended cultures are mixed. Immobilization has also dimpled metal sheets for continuous algae attachment and harvest- been shown to result in enhanced hydrocarbon production (Bailliez ing. They qualitatively showed that more algae are attached to a 692 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702

Table 4 Algae biomass production and wastewater nutrient removal in algal bioﬁlm systems.

Design Nutrient loadinga (mg l− 1 day− 1) Nutrient removal Biomass production (g m− 2 day− 1) Scale Reference

PVC brushes TN: 5.5 TN: 87% Not reported Lab Wei et al. (2008) TP: 1.7 TP: 98% Rotating styrofoam disks N: 45–180 N: 100% 2.2 Lab Przytocka-Jusiak et al. (1984) P: 1.7–3.3 Rotating aluminum disks N: 312 N: 60% Not reported Bench Torpey et al. (1971) Polycarbonate ﬂow lanes P: 1.2 P: 100% 2.9 Lab Guzzon et al. (2008) Algal turf scrubber TN: 160–1030 TN: 36–92% 5.3–5.5 Bench Wilkie and Mulbry (2002) TP: 80–160 TP: 51–93% Polystyrene rocker system N: 30.9 N: 100% 2.59 Lab Johnson and Wen (2010) P: 1.8 P: 70%

a Soluble/dissolved forms of N and P unless specified as Total N (TN) and Total P (TP). textured steel surface than to a smooth steel surface. Johnson and An attached process could also be economical for wastewater Wen (2010) compared the performance of an attached culture to a treatment. An algal turf scrubber in Florida had a cost of $24 per kg of suspended culture grown under the same conditions and reported phosphorus removed, while an engineered wetland process had a cost greater yields from the attached culture and the same lipid content. of $77 per kg phosphorus removed (Sano et al., 2005). Another study The attached culture was grown on a section of submerged places the cost of phosphorus removal from dairy wastewater at polystyrene operated using a rocking motion. $31.10 per kg (Pizarro et al., 2006). Phosphorus removal costs using Another design, the Algal Turf Scrubber, consists of a plastic mesh conventional chemical precipitation and/or non-algal biological for filamentous algae attachment with intermittent wave surges. It has removal vary depending on plant conditions. From cost simulations been reported to have a biomass production of 15–27 g m−2 day−1 by Jiang et al. (2004), an estimated total annual cost for a 10 MGD (Adey et al., 1993). Several other studies with this design have shown plant with an influent phosphorus concentration of 7.5 mg l− 1 and an good nutrient uptake and biomass productivity that typically ranges effluent concentration of 2 mg l− 1 can be calculated as approximately from 5 to 20 g m−2 day−1 (Mulbry and Wilkie, 2001; Mulbry et al., $105 per kg phosphorus removed. Keplinger et al. (2004) analyzed six 2005; Wilkie and Mulbry, 2002). The filamentous algae grown on the small Texas treatment plants with a combined permitted discharge of Algal Turf Scrubber has low fatty acid content, however, reducing its 4.7 MGD. From their data an average cost of $87 per kg phosphorus value as a biofuel feedstock (Mulbry et al., 2008). Table 4 summarizes removed by alum-based precipitation can be calculated. algal biofilm designs with respect to nutrient loading and removal, biomass productivity, and scale. 4. Algae harvesting methods

3.3. Algae production cost Current harvesting methods include chemical based, mechanical based, and to a lesser extent, electrical based operations, with Cost estimates for production of algae with suspended growth various combinations or sequences of these methods also common systems vary widely, and there is not much available information on (Bernhardt and Clasen, 1991; Danquah et al., 2009; Kumar et al., the cost of producing algae using attached growth systems. Table 5 1981). Biological based methods are also being investigated as a summarizes costs and cost estimates for different types of production cost reducing means of harvesting. There is no proven single best systems, including future cost estimates assuming certain engineering method of harvesting microalgae (Shelef et al., 1984). improvements are realized. Care should be taken when comparing these values across studies, as different parameters and assumptions 4.1. Chemical based were used. Nevertheless, with an estimated algae production cost of $0.70–$0.97 kg− 1 for an algal turf scrubber, it appears that an Because of the small size of algae cells, chemical flocculation is attached system may be a good option for low cost algae production, often performed as a pretreatment to increase the particle size before although more research is needed in this area. A very basic economic using another method such as flotation to harvest the algae. analysis by Johnson (2009) also placed the capital and operation costs Electrolytes and synthetic polymers are typically added to coagulate of his attached growth system at or below those of a conventional (neutralize charge) and flocculate the cells, respectively (Bernhardt raceway pond. and Clasen, 1991). Because of the +3 charge of the aluminum and

Table 5 Algae production costs and cost estimates.

Type Production method Cost ($kg− 1) Notes/Assumptions Reference

Cost Open pond 5 Spirulina production Benemann (2008) Cost Open pond 3.60a Dunaliella production Brennan and Owende (2010) Estimate Attached culture 0.70–0.97 Using dairy wastewater Pizarro et al. (2006) Estimate Open pond 6.93a Netherlands location Norsker et al. (2011) Estimate Closed PBR 5.81a Netherlands location Norsker et al. (2011)

Estimate Open pond 3.80 Free CO2 Chisti (2007)

Estimate Closed PBR 2.95 Free CO2 Chisti (2007) a Forward-looking estimate Open pond 1.79 Free CO2 and growth media, 60% improved Norsker et al. (2011) photosynthetic efﬁciency, Dutch Antilles location a Forward-looking estimate Closed PBR 0.98 Free CO2 and growth media, 60% improved Norsker et al. (2011) photosynthetic efﬁciency, Dutch Antilles location Forward-looking estimate Open pond 0.60 100× increased production for better economy of scale Chisti (2007) Forward-looking estimate Closed PBR 0.47 100× increased production for better economy of scale Chisti (2007)

a Calculated using a conversion factor of 1.4 dollars per euro. L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 693 ferric cations, aluminum sulfate and ferric chloride are often used for a solids concentration of 6.3%. At such concentrations, any additional charge neutralization. When considering downstream processes to harvesting or concentrating operation is likely unnecessary. produce bioproducts from algae, the use of metal salts for coagulation Table 6 compares the most common mechanical harvesting and flocculation is cautioned. Aluminum and sulfate have been shown methods for algae with regard to benefits, limitations, solids recovery, to inhibit the specific methanogenic activity of methanogenic and and solids concentrations. acetogenic bacteria fed wastewater sludge (Cabirol et al., 2003). Land application of aluminum treated sludge can increase heavy metal 4.3. Electrical based uptake and cause phosphorus deficiencies in plants (Bugbee and Frink, 1985). Separation methods based on electrophoresis of the algae cells have Natural polymers that do not involve the same concerns of also been attempted. Because of the negative charge of algae cells, they secondary pollution may also be used as flocculants, although these can be concentrated by movement in an electric field (Kumar et al., are less studied. Divakaran and Sivasankara Pillai (2002) saw successful 1981). The major benefit of approaches based on these principles is that flocculation and settling of algae by adding chitosan. Cationic starch has no chemical addition is required; however, the high power require- also been identified as an effective flocculating agent (Pal et al., 2005), ments and electrode costs do not make for an appealing harvesting and has been shown to flocculate freshwater microalgae in jar test method, especially for large-scale applications (Uduman et al., 2010). experiments (Vandamme et al., 2009). 4.4. Biological based

4.2. Mechanical based Algae are known to sometimes flocculate spontaneously without chemical addition (Sukenik and Shelef, 1984). Exploiting and Centrifugation is perhaps the most rapid and reliable method of controlling this ability could significantly reduce harvesting costs. recovering suspended algae. Centrifugal forces are utilized to separate Although the terms are used somewhat interchangeably, autofloccu- based on density differences. According to Shelef et al. (1984), nozzle lation and bioflocculation describe different phenomena. type disc centrifuges are easily cleaned and sterilized and are suitable Autoflocculation occurs at high pH levels caused by consumption for all types of microalgae, but the high investment and operating of dissolved carbon dioxide. Increasing pH causes supersaturation of costs must also be considered. The U.S. Department of Energy has calcium and phosphate ions. If an excess of calcium ions is present, the concluded that at the current level of centrifugation technology, this calcium phosphate precipitate will be positively charged. Algae cells method is cost-prohibitive for any large scale use (U.S. DOE, 2010). serve as a solid support for the precipitant and charge neutralization is Low-cost filtration methods are often used to harvest filamentous accomplished (Lavoie and de la Noüe, 1987). Autoflocculation may algae strains (Vonshak and Richmond, 1988). Wood (1987) described not be possible in all waters. Sukenik and Shelef (1984) determined a high rate algae pond system to select for more easily harvested that optimum concentrations for calcium phosphate precipitation and filamentous algae by microscreening to retain larger cells and autoflocculation at a pH of 8.5–9 were 3.1–6.2 mg l− 1 phosphate and washing out smaller non-filamentous algae. Other researchers, 60–100 mg l− 1 calcium. Nurdogan and Oswald (1995) overcame such however, have not been able to confirm dominance of these species a limitation by adding lime to a raceway pond. This alone brought (Hoffmann, 1998), and for applications in biofuels, filamentous algae nitrogen, phosphorus, and algae removal to above 90%. are less useful due to their low lipid content (Mulbry et al., 2008). For The term bioflocculation is usually meant to describe flocculation smaller suspended algae, tangential flow filtration is considered to be caused by secreted biopolymers. Sedimentation of phytoplankton more feasible than dead-end filtration, but membrane fouling and blooms has been positively correlated with an increase in EPS replacement are significant costs (Uduman et al., 2010), and power concentrations (Bhaskar and Bhosle, 2005). Passow and Alldredge requirements are high (Danquah et al., 2009). (1995) reported that a controlled diatom bloom underwent mass Sedimentation is a low cost harvesting option that can typically give flocculation soon after a sudden increase in the amount of cells concentrations of 1.5% solids (Uduman et al., 2010), but because of the enclosed by biopolymer. EPS produced by algae biofilms in a trickling fluctuating density of algae cells, reliability is also low (Shen et al., filter enhanced solids flocculation in a later clarifier operation (Shipin 2009). At settling rates of 0.1–2.6 cm h-1, sedimentation is relatively et al., 1999). EPS production has been reported to be maximal at the slow, and much of the biomass may deteriorate during the settling time end of the growth phase (Bhaskar and Bhosle, 2005; Staats et al., (Greenwell et al., 2010). 1999), although light and temperature conditions also affect bio- Dissolved air flotation (DAF) is a method commonly used in flocculation (Wolfstein and Stal, 2002). wastewater treatment sludge removal (Friedman et al., 1977). In Another biological approach is microbial flocculation of algae. Lee algae rich waters, DAF is usually preferred over sedimentation et al. (2008) added flocculating microbes to an algae culture. After methods (Teixeira and Rosa, 2006). The major advantage of DAF is feeding 0.1 g l− 1 acetate, glucose, or glycerin and mixing for 24 h, that it has been proven at large scales, but the use of flocculants can be they achieved 90% recovery and a concentration factor of 226. Oh et al. a problem in downstream processing of the algae (Greenwell et al., (2001) reported better efficiency using a flocculant from soil microbes 2010; Hoffmann, 1998). than with aluminum sulfate or polyacrylamide for harvesting Chlorella Designs that use attached algae biofilms also mechanically harvest vulgaris. the algae. Filamentous algae grown on a turf scrubber could be Another biological based approach to harvesting involves the use vacuumed (Jensen, 1996) or scraped (Adey, 1982, 1998). Johnson and of planktivorous fish such as tilapia. The Controlled Eutrophication Wen (2010) used simple scraping to harvest a Chlorella biofilm that had Process starts with raceway ponds to grow algae. The algae are then

Table 6 Comparison of mechanical harvesting methods for algae. Adapted from Shelef et al. (1984), Shen et al. (2009), Greenwell et al. (2010), and Uduman et al. (2010).

Method Solids concentration after harvesting Recovery Scale Major beneﬁts Major limitations

Centrifugation 12–22% N90% Bench Reliable, high solids conc. Energy intensive, high cost Tangential filtration 5–27% 70–90% Bench Reliable, high solids conc. Membrane fouling, high cost Gravity sedimentation 0.5–3% 10–90% Pilot Low cost Slow, unreliable Dissolved air flotation 3–6% 50–90% Pilot Proven at large scale Flocculants usually required 694 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 batch fed to caged fish, and the fish droppings and any sedimented are lysed by cavitation bubble collapse (Larach, 2010). There is no algae are brought to the surface on an inclined conveyer belt to be fed mention of any wastewater treatment applications. to an anaerobic digester (Brune et al., 2007). Rectenwald and Drenner Seambiotic is an Israeli company growing algae in outdoor

(2000) described a similar process of passing nutrient rich water raceway ponds near power plants. Concentrated CO2 from flue gasses through porous screens to grow periphyton. Excrement from tilapia is fed to the raceway ponds (Weiss, 2008). Wastewater treatment feeding on the algae is collected in a sediment trap. Reductions in total applications are not discussed. phosphorus and total nitrogen of 82% and 23%, respectively, were General Atomics has several patents related to algae cultivation. observed. Dunlop and Hazlebeck (2010) explain the use of submerged horizontal bars in a growth channel to produce vortices in the passing 5. Approaches to algae production and harvesting in industry liquid. This is intended to improve vertical mixing for better light distribution through the culture. Wastewater treatment is not Because of the high commercial potential of algae based biofuels and discussed. algae based wastewater treatment, research and development of algae Blue Marble is attempting to specialize in the anaerobic digestion production and harvesting technologies are being conducted by private of algae and other biomass to produce biomethane and ammonia companies and industries. Many of the needed innovations can be fertilizer (Stephens, 2010), although earlier patent applications solved through collaborations between academia, algae production describe a production and harvesting device. The device is made fl companies, the wastewater treatment industry, and users of algae- from a micron mesh liner attached to a buoyant frame oated on an based technologies including municipalities and industries. Table 7 lists open body of water. The liner is intended to allow water and nutrients algae production and harvesting designs and processes, along with in without letting cells out. The buoyancy of the frame is controlled by scale of application, associated companies, and involvement with adjusting the amounts of water and air in the frame tubing. After fi wastewater treatment. Table 7 is grouped according to production suf cient algae growth, the buoyancy can be increased to lift the approach and ordered according to scale. It is not intended to rank or entire apparatus out of the water for collection of the culture endorse the companies in any way. (Stephens et al., 2009a). A related application describes the fl For the purposes of this section, scale is defined as laboratory if potential of using the oating pond reactors to remove undesirable volumes of less than 10 gal are used, bench at 10 to 1000 gal, pilot for components such as nitrogen and phosphorus from water (Stephens several thousand gallons or a site of 0.5 to 10 acres, demonstration for et al., 2009b). a site of 20 to 80 acres or a flow of approximately 1 MGD, and full for a site greater than 80 acres or if flow is several MGD. 5.1.2. Closed reactors Solix Biofuels and A2BE Carbon Capture are assignees on a patent application describing a closed reactor system with a rotatable 5.1. Reactor designs for algae production internal transparent insulator (Sears, 2007). The insulator can be placed between the bulk of the reactor and the air, leaving thermal Several companies are seeking to increase algae production contact with the ground, or the insulator can be placed between the through reactor design. Most reactors fall under the category of bulk of the reactor and the ground, leaving thermal contact with the open ponds or closed reactors, though some are best described as a surrounding air. The reactor design also contains a harvesting hybrid combination of the two. Hybrid designs attempt to balance the chamber where fluid motion maintains a whirlpool to pre-concen- fi bene ts of low cost open ponds with the control of a closed system. trate the algae before it is passed through a roller press. Wastewater This is usually accomplished by placing a cover over an open pond or treatment is not directly discussed, but the patent application does fi channel. A smaller number of designs are for algal bio lm attached mention that the algae can be largely fed by industrial, agricultural, growth. and municipal waste products. Solix has other designs including floatable vertical tubular reactors for improved thermal regulation 5.1.1. Open ponds (Willson et al., 2008), and a tubular reactor that incorporates gas

Many of the companies that have been longest involved in the permeable membranes into sections of the tubes to improve O2 mass production of algae grow and harvest the filamentous release (Willson et al., 2009). cyanobacteria Spirulina as a nutraceutical product in clean, non- Sunrise Ridge Algae also claims to have a low cost tubular reactor wastewater systems. Earthrise Nutritionals and Cyanotech are two design made of flexible materials that can be rolled out on site and companies using open raceway ponds to grow Spirulina (Jensen and mixed by air sparging (Whitton, 2008). This particular patent Reichl, 1997). Because of the filamentous morphology of Spirulina, application does not discuss any uses for wastewater treatment, but harvesting through simple filtration methods is effective (Vonshak like Petroalgae, the company's recent focus appears to be on and Richmond, 1988). wastewater treatment using duckweed. Petroalgae is one company using the open pond approach, Algae Systems is a company that has licensed NASA's Offshore although the end product is not technically microalgae. The company Membrane Enclosure for Growing Algae (OMEGA) system in addition is listed as assignee on a patent application describing a central seed to purchasing intellectual property from Greenfuel Technologies (no area with several final ponds radiating from the central area (Howard longer in operation). The OMEGA system consists of flexible plastic et al., 2008). The application states that wedge shaped ponds are bags that are at least partially permeable to CO2 and O2. The bags are useful for growing algae continuously because the inoculum can be filled with domestic wastewater and placed in seawater. The idea is added at the point of the wedge so that as the culture moves toward for the reactors to automatically dewater as the treated wastewater the wide section, there is greater surface area for sunlight and leaves through forward osmosis (Trent et al., 2010). Greenfuel multiplying cells. No application to wastewater treatment is men- Technologies had a portfolio containing several reactor designs; one tioned. Despite the company name, it appears that Petroalgae is describing a closed reactor designed to float on a pond for better currently producing duckweed, not microalgae. Javan et al. (2010) thermal regulation (Berzin et al., 2009), and another describing a describe a paddlewheel-mixed raceway for growing Lemna. Harvest- modified air lift bioreactor (Berzin and Wu, 2007). ing is accomplished by lowering a conveyer belt or surface skimmer Origin Oil is also designing reactors that can better distribute light into the raceway before transporting the Lemna to a screw auger. throughout the culture. A perforated rod is placed in the middle of the

Kai Bioenergy is another company using the open raceway pond reactor. Nutrients and CO2 are delivered through the perforations. approach. Foam fractionation is used to concentrate cells before they Light is channeled through the rod to transparent paddles connected Table 7 Companies involved in algae production and/or harvesting.

Production approach Harvesting approach Company Scalea Reference 686 (2011) 29 Advances Biotechnology / Sims R. Christenson, L.

Open ponds Raceway ponds Foam fractionation, cavitation bubble disruption Kai Bioenergy Not disclosed Larach (2010) Floatable pondb Pond lifted out of water Blue Marble Energy No longer producing algae Stephens et al. (2009a, 2009b) Open ponds Flocculation and DAF Honeywell's UOP Bench Marker et al. (2009) Two stage process: CSTR feeds an unlighted PFRb Vacuum belt Algae to Energy (A2E) Pilot Shepherd (2010) Two stage process: CSTR to PFR Flocculation then settling or DAF General Atomics Small pilot (6000 gal pond), Dunlop and Hazlebeck (2010), Hazlebeck and Dunlop (2008, 2010), developing a 40 acre site Raceway ponds Autoflocculation, centrifugation Seambiotic Pilot (1/2 acre site) Weiss (2008) Raceway ponds Flocculation then settling or DAF Aurora Algae Pilot (1 acre site) Vick and Fleischer (2009), Vick (2010), Weissman et al. (2010), Weissman and Radaelli (2010) Clay raceway ponds Gravity settling followed by other Aquatic Energy Pilot (2 acre site) Demaris et al. (2009) followed by starvation pond Two stage process: closed reactors to open ponds Gravity settling followed by centrifugation HR Biopetroleum Pilot (6 acre site) Huntley and Redalje (2010) Raceway pondsc Conveyer belt or skimmer PetroAlgae Demonstration (40 acre site) Javan et al. (2010) Raceway ponds Cell-viable extraction Phycal Demonstration (40 acre site) Swanson et al. (2010), Lane et al. (2010) Open pondsb Planktivorous fish LiveFuels Demonstration (45 acre site) B. Wu et al. (2010a, 2010b) Raceway ponds for Spirulina Filtration Cyanotech Full (90 acre site) Jensen and Reichl (1997) CEP (raceway ponds)d Inclined conveyer belt for fish feces Kent BioEnergy Full (160 acre site) Brune et al. (2007), Schwartz et al. (2010)

Closed reactors – Tubular reactorsb Not speciﬁed A2BE Carbon Capture Bench Sears (2007) 702 NASA's OMEGA systemd Forward osmosis Algae Systems Bench Trent et al. (2010) Flat panel or tubular reactorsb 3rd party Bionavitas Bench Wilkerson et al. (2009), Wilkerson and Watters (2009), Closed reactors with internal light rodsb Cavitation bubble disruption then skimming Origin Oil Reactor = bench; Eckelberry and Eckelberry (2009) Extraction method = pilot − (300 gal min 1 )

(continued on next page) 695 696

Table 7 (continued) Production approach Harvesting approach Company Scalea Reference .Crsesn .Sm itcnlg dacs2 21)686 (2011) 29 Advances Biotechnology / Sims R. Christenson, L. Tubular reactors Centrifuge with textured walls Scipio Biofuels Bench Wells and Snyder (2010) Tubular reactors Not specified Sunrise Ridge Algae Bench Whitton (2008) Helical tubular reactors Not specified Texas Clean Fuels Bench Gal (2009) Corrugated panel reactore Not specified Joule Unlimited Bench Devroe et al. (2009, 2010), Van Walsem et al. (2010) Closed greenhousese No (secreted ethanol) Algenol Pilot Woods et al. (2010) Bag reactors with light Induced flocculation Sapphire Energy Pilot Fang et al. (2010), Mendez et al. (2009a, 2009b, 2010a, 2010b), delivery rodse Olaizola (2010) Tubular reactorsb Whirlpool concentrator then centrifuge Solix Biofuels Pilot (2 acre site) Willson et al. (2008, 2009)

Hybrid designs Covered raceway ponds Concentrate to 10–20% slurry Genifuel Not disclosed Oyler (2008a, 2008b, 2010) Covered ponds Evodos centrifuge MBD Energy Small pilot (1000 gal pond) Boele (2010) Rapid algae farms (covered ponds)b Capillary extraction belt Algaeventure Systems Pilot Youngs and Cook (2010) Simgae system (covered Flocculation Diversiﬁed Energy Demonstration (40 acre site) Keeler et al. (2010) furrows)b

Biofilm reactors Biofilms on polyester sheets Sprayed with water jets Greenshift Pilot Bayless et al. (2003) Biofilms in open channelsd Sprayed with water jets SBAE Industries Pilot J. Vanhoutte and K. Vanhoutte (2009) − Biofilms on baffled rotating contactorsd Collect sheared biofilms Algaewheel Pilot (100,000 gal day 1 ) Limcaco (2010) Turf scrubber for filamentous algaed Vaccum or mechanically scrape turf Aquafiber Technologies Full (7.5 MGD) Jensen (1996) d – Turf scrubber for filamentous algae Mechanically scrape turf Hydromentia Full (up to 30 MGD) Adey (1982, 1998) 702

Other Not speciﬁede No (secreted fatty acids and alcohols) Synthetic Genomics Bench Roessler et al. (2009, 2010) Heterotrophic fermentation Not speciﬁed Solazyme Demonstration scale fermentation Dillon (2008)

a According to information available on company website. b Possible applications to wastewater treatment mentioned. c Duckweed product is not technically microalgae. d Demonstrated wastewater treatment or specifically intended for wastewater treatment. e Genetically modified algae. L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 697 to the ends. The rod and paddles also act as a static mixer. Cell biomass is then recovered after settling (J. Vanhoutte and K. disruption is achieved using cavitation (Eckelberry and Eckelberry, Vanhoutte, 2009). 2009). A related patent application mentions that, although not an Greenshift Corp. has rights to a gas treatment reactor made of exemplary use, the light arrays could be incorporated into wastewater vertical polyester panels for attached algae growth. Optical wave- aeration tanks (Shigematsu and Eckelberry, 2009). The company has guides can be placed between each panel to distribute light to each announced that it has filed a patent application for an attached algae side. Harvesting is done by increasing the pressure of the water system for wastewater treatment, but the application is not yet delivery system to spray the biofilms off the panels (Bayless et al., published. 2003). GS Clean Tech, a subsidiary of Greenshift, is the assignee on an Bionavitas is a company attempting to overcome the challenge of application that describes the use of a similar system in conjunction PAR delivery by collecting solar radiation and delivering it to a plurality with an ethanol production plant (Winsness et al., 2007). This biofilm of optical waveguides spaced within the reactor to more efficiently reactor was designed to treat waste gas streams, but it may not be distribute the light (Wilkerson and Watters, 2009; Wilkerson et al., easily adapted to wastewater treatment facilities. 2009). The patent applications discuss the possibility of using wastewater effluent as part of the nutrient supply system to the reactor. 5.2. Harvester designs and harvesting processes Texas Clean Fuels uses a basic helical tubular reactor design. A transparent cylinder is used as the core to which the tube is wrapped To overcome the challenge of harvesting suspended algae, around so that light can reach both sides of the cylinder (Gal, 2009). industry researchers are looking for improvements to harvester The patent application does not mention wastewater treatment. designs and/or processes. Some companies are attempting to improve mechanical harvesters or create new ones while others are focusing on biological based harvesting. A few companies are attempting to 5.1.3. Hybrid designs bypass the algae separation step altogether. Diversified Energy Corp. has created the Simgae system for producing algae. The approach is to make the setup and operation 5.2.1. Mechanical harvesters of algae reactors as simple as possible by designing them so much of Algae to Energy, or A2E, uses what it calls the Shepherd Harvester the work can be done using typical farm equipment. Furrows are lined for algae separation (Shepherd, 2010). The harvester uses a with plastic, filled with media, and covered (Keeler et al., 2010). continuous belt that moves through the algae culture and a vacuum Harvesting can be done at the end of the furrows after sufficient system. As the belt moves, any algae collected on the belt is harvested growth has occurred. According to the patent application, at least a by the vacuum system before the belt passes through the culture portion of the fertilizer solution fed to the furrows may come from again. The patent application does not directly discuss use of the dairy farms and wastewater treatment facilities. harvester in wastewater treatment plants, but the need to incorporate Genifuel Corporation's reactor design is also a hybrid system. Oyler large scale algae cultivation into existing infrastructure such as (2008a) describes a covered paddlewheel mixed raceway with sewage treatment facilities is mentioned. continuous gas injection to keep a positive pressure in the chamber Algaeventure Systems, Inc. also uses a continuous belt harvester to prevent inflow and contamination from the outer environment. based on capillary extraction (Youngs and Cook, 2010). The design Wastewater treatment is not discussed. uses a primary belt to collect algae and a secondary capillary belt made of a super absorbent polymer. The secondary belt is in contact 5.1.4. Biofilm reactors with the bottom portion of the primary belt so that water is pulled Except where a genetically modified culture or other monoculture through the algae and primary belt into the secondary belt. The dried is intended, most algae production designs could be tailored to handle biomass on the primary belt is collected and the secondary belt is wastewater as a nutrient source. There are several approaches, compressed to drain water before it contacts the primary belt again. however, that are specifically intended to be incorporated into The patent application does not discuss the use of the harvester in wastewater treatment, and these are most often biofilm based wastewater treatment, but the company does discuss the potential designs such as those discussed in this section. use of wastewater in covered ponds called Rapid Algae Farms on their Hydromentia has rights to the Algal Turf Scrubber. Filamentous website. General Atomics has also awarded a purchase order to algae grow on a plastic mesh in a spillway as wastewater or other Algaeventure Systems for their harvesting device. nutrient rich water surges over the surface (Adey, 1982, 1998). MBD Energy is an Australian company using coal plant wastewater Mature turf can be harvested by pulling a scraper behind an ATV and covered raceway ponds for algae production. The company is (Stewart and Zivojnovich, 2003). Aquafiber Technologies Corporation collaborating with Evodos, a Dutch company, and using their uses a similar approach with a vacuum harvester to obtain the mature separators. The Evodos separator is a centrifuge that allows for easier turf (Jensen, 1996). Algaewheel Technologies uses a modified rotating removal of solids after concentration. The inner assembly is made of biological contactor design to grow algae and treat wastewater. The curved but flexible disks. This inner assembly can be removed and contactors are much smaller and are baffled so that air jets can rotate rotated so that the curved disks become straight and solids become them. The interior of each contactor is filled with polystyrene balls to unwedged (Boele, 2010). support bacterial growth while algae biofilms grow on the outer Scipio Biofuels grows algae in closed tubular reactors. Their baffles in a symbiotic relationship (Limcaco, 2010). continuous harvester is basically a low speed centrifuge. A circular SBAE Industries, from the Netherlands, is another one of the few chamber with a textured side wall rotates to force cells against the companies working on biofilm based algae production. K. Vanhoutte side wall. Because flocs or larger cells cannot pass over the rough and J. Vanhoutte (2009) describe a conveyer belt system where a surface as readily as smaller cells, they remain against the wall. A growth substratum is partially submerged in wastewater. A contin- skimmer blade then continually passes along the wall to remove these uous operation can be developed by starting growth at a point farthest flocs (Wells and Snyder, 2010). The patent application does not from a central collection area and allowing a certain amount of time discuss any wastewater treatment capabilities. for growth before reaching the harvesting area. SBAE's Diaforce system consists of sections of growth substrata placed in an open 5.2.2. Biological based harvesting channel with wastewater flowing through. As biofilms become Kent Bioenergy has rights to the Controlled Eutrophication Process established, sections are removed and taken to a harvesting developed at Clemson that was described in Section 4.4 of this area where the biofilm is removed by spraying with water jets. The document. The company is also the assignee on a patent application 698 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 describing a sequence of decanting operations to select for a culture with low chlorophyll content so light can reach deeper into the more disposed to flocculate and settle. Flow in a raceway pond is culture. 2-hydroxy-5-oxoproline is also added to enhance growth stopped and a settling period elapses. An upper layer of water is then (Vick and Fleischer, 2009). To maintain selectivity, glyphosate removed along with any algae in it. The removed volume is then herbicide can be added to glyphosate resistant Nannochloropsis replaced and the process is repeated until sediment-ready algae cultures (Vick, 2010). They also report that Nannochloropsis will sufficiently dominate the culture (Schwartz et al., 2010). The patent better dominate a lower salinity environment and recover more application specifically describes the use of such a process for quickly from disinfectant exposure than invasive strains (Weissman wastewater treatment, and the technique was demonstrated in two and Radaelli, 2010), and that ozone shock can be used for the same treatment ponds measuring 80 ft2 each. purpose (Weissman et al., 2010). None of the documents discuss any Live Fuels Inc. is another company utilizing fish as a means of potential wastewater treatment applications, and the company's harvesting algae. The planktivorous fish, such as tilapia, are harvested focus on monocultures of Nannochloropsis would be incompatible for oil and fishmeal (Wu et al., 2010a). A series of foam fractionation with the mixed culture constraint of a wastewater treatment lagoon. units may be used to pre-concentrate the algae as well (Wu et al., Cellana is an algae biofuels company originally created as a joint 2010b). Regarding wastewater treatment, the patent applications venture between HR Biopetroleum and Royal Dutch Shell, though it is briefly mention the possibility of using agricultural, industrial, or now owned solely by HR Biopetroleum. HR Biopetroleum is the municipal wastewater in the system. Live Fuels also has a separate assignee on a patent that describes a continuously operated system of Patent Cooperation Treaty application describing the use of transgenic closed reactors used to inoculate batch operated open ponds (Huntley fish in this process (Stephen and Morgenthaler, 2010). and Redalje, 2010). The idea is to prevent contamination of the open For Sapphire Energy, Mendez et al. (2009a) describe algae ponds by ensuring the inoculum from the closed reactors is enough to genetically modified to enable controlled flocculation and simpler give the preferred organism an advantage. There is no indication that harvesting. The algae are modified to express a ligand or receptor Cellana is looking to apply their technology to wastewater treatment. molecule such as an antibody or antigen. The molecule can be attached to the cell wall or secreted. For example, a culture expressing an 5.3.2. Lipid accumulation antibody could be mixed with a separate culture expressing the Aquatic Energy uses an additional production stage after sufficient corresponding antigen to induce flocculation. Expression of a ligand/ growth has been achieved in clay lined raceways. After the raceways, receptor pair could be sequentially induced to initiate flocculation. cells enter a secondary stress pond for nitrogen starvation and lipid accumulation for 48 h before being harvested (Demaris et al., 2009). 5.2.3. Bypassing the harvesting step For Genifuel Corporation, Oyler (2008b) describes a two-stage Phycal is trying to bypass much of the harvesting and dewatering process for producing algae with high lipid content consisting of a first process by performing non-destructive oil extraction of live cells. stage of autotrophic conditions to produce the biomass and a second Phycal's process involves mixing a portion of a Nannochloropsis stage of heterotrophic conditions to increase lipid content. culture with a lipid extracting solvent such as dodecane for Before the live extraction described in Section 5.2.3, Phycal's approximately 5 min while sonicating the cells at 40 kHz for 2 s process increases lipid production by inhibiting nitrate uptake, either (Swanson et al., 2010). This process also aids in reducing levels of through the addition of chlorate or by inducing the production of a predators and unwanted species (Lane et al., 2010). The possibility of nitrate reductase inhibitor in a genetically engineered culture using wastewater is not discussed, and it would likely be difficult if (Swanson et al., 2010). monocultures of Nannochloropsis are intended. Algenol Biofuels Inc. is also attempting to bypass the biomass 5.3.3. Nutrient and gas recycle harvesting step altogether by using genetically modified algae or GS Clean Tech's patent application for a vertical sheet biofilm cyanobacteria capable of secreting ethanol. Such a culture would be system described in Section 5.1.4 also describes the use of CO2 from an enclosed in greenhouse where evaporated water and ethanol would ethanol production plant to grow algae. The algae biomass is then condense on the ceiling and travel to a collection trough (Woods et al., added to the original feedstock of the ethanol plant, thus recycling the

2010). CO2 byproduct of ethanol fermentation (Winsness et al., 2007). Synthetic Genomics, partnered with ExxonMobil, is using genetic For General Atomics, Hazlebeck and Dunlop (2008) have described engineering approaches to create algae for fuel production. Roessler et al. a gas–liquid contactor reactor used to scrub CO2 before feeding the (2009) describe genetically modified algae or cyanobacteria capable of solution to an algae culture. Greenfuel Technologies also had an secreting fatty acids into the growth media. The fatty acids can then be application describing a gas–liquid contactor to scrub flue gas before collected by liquid–liquid extraction or chromatography. Another patent feeding the liquid to a photobioreactor (Wu et al., 2007). Another application deals not with fatty acids, but with secreted branched chain General Atomics patent describes the recycling of nutrients back to a alcohols (Roessler et al., 2010). growth chamber after cell lysing and transesterification steps. More specifically, the non-oil fraction of lysed cell matter from the lysing 5.3. Process design step is combined with the glycerin byproduct of the transesterification step before being fed to a chemostat for algae growth (Hazlebeck Approaches to improved process design often have the objectives and Dunlop, 2010). of greater culture control, increased cellular lipid content, or cost Genifuel Corporation has a patent application describing the reduction through nutrient and gas recycle and the utilization of gasification of wet biomass before recycling the CO2 and nutrients waste streams. back to a growing chamber (Oyler, 2010). Honeywell's UOP is the

assignee on a patent application describing the capture of CO2 from a 5.3.1. Culture control biodiesel production process. The CO2 is then fed to an algae culture to Aquatic Energy claims to maintain culture selectivity simply by produce more biomass for the process (Marker et al., 2009). matching the residence time of their clay lined raceway ponds to the doubling time of their target organism (Demaris et al., 2009). No 5.4. Genetic manipulation applications to wastewater treatment are discussed in the patent application. Algenol Biofuels and Synthetic Genomics are using genetic Aurora Algae, previously called Aurora Biofuels, has patent engineering approaches to enable secretion of ethanol, fatty acids, literature describing the use of mutant pale-green Nannochloropsis or alcohols as described in Section 5.2.3.InSection 5.2.2, the Sapphire L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 699

Energy method of controllable flocculation using genetically modified treatment into their production approaches. Although few companies algae is described. are operating beyond the small pilot scale, there is more wastewater Sapphire Energy has several other patent applications related to integration with companies at demonstration and full scale operation, the genetic manipulation of algae. Mendez et al. (2009b) describe the likely because cost effective scale-up beyond small pilot plants construction of synthetic chloroplasts and Mendez et al. (2010a) necessitates the use of such available resources. describe the increased expression of fatty acid synthesizing enzymes Fig. 2 shows the number of companies reviewed in this paper in algae. On the production side, Olaizola (2010) describes the use of operating at different scales and the proportion of those companies transparent rods with floats on top and weights on the bottom in an using open ponds, closed reactors, hybrid designs, or biofilm reactors. algae pond or reactor. Any light that enters the rods is delivered to the The majority of companies using a closed reactor approach are darker portions of the culture below the surface. Sapphire also has operating at bench scale, and no closed reactor approach is operating patent cooperation treaty applications for genetically modified beyond the small pilot scale. This is likely due to the difficulty in herbicide resistant algae (Fang et al., 2010), and genetically modified scaling up closed reactors relative to other production approaches. At salt tolerant algae (Mendez et al., 2010b). pilot, demonstration, and full scales, the most common approach is Joule Unlimited is focused on creating enhanced algae through open ponds, although at full scale, the attached algal turf scrubber genetic engineering. Devroe et al. (2010) describe the upregulating, represents two of the four operations. downregulating, or knocking out of specified genes in order to potentially give enhanced light utilization, carbon fixation, NADH and 6. Conclusions NADPH production, thermotolerance, pH tolerance, salt tolerance, flue gas tolerance, nutrient independence, and near infrared absorbance. Much of the research addressing algae production and harvesting Devroe et al. (2009) disclose mechanisms to confer photosynthetic is currently confined to the laboratory. Although many companies properties to a heterotrophic organism with better understood have moved from laboratory and bench scales to small pilot scale, but techniques for genetic manipulation and industrial processing such the major challenges of nutrient supply, land and water availability, as Escherichia coli. Joule's reactors are modified flat panel closed PAR delivery, gas exchange, environment control, and culture reactors with corrugated panels to act as static mixers for increased integrity have limited further scale-up, and the number of studies fluid turbulence (Van Walsem et al., 2010). and companies operating at demonstration and full scale is limited. Solazyme is best known for producing algae under heterotrophic Overcoming current challenges to the production and harvesting conditions using fermentation technology, but a patent application of algae will benefit both the biofuel and wastewater treatment fields. from earlier work describes genetic alterations to algae to down- It appears, however, that this collaborative potential has not been regulate production of light harvesting pigments so more light can realized, as those testing algae production systems have not often pass the top layer of cells and reach the bulk of the culture (Dillon, integrated their research with the wastewater industry's need for 2008). algae production technologies. Using wastewater as a resource and combining wastewater treatment with the production of algae based 5.5. Summary of approaches in industry bioproducts can overcome several of the major challenges identified herein. Additionally, the existing infrastructure of wastewater Fig. 1 shows the number of algae production companies reviewed treatment facilities can be utilized for managed algae production, in this paper operating at bench, pilot, demonstration, and full scale, thereby reducing capital costs and scalability challenges, providing a as well as the proportion of those companies that have discussed the unique training ground for the development of commercial scale algae potential of integrating wastewater treatment resources or have production. Despite these benefits, only a few preliminary studies designed for and/or demonstrated it. Although several companies have been conducted to produce biofuels and bioproducts from algae have moved from bench scale to small pilot operations, the majority of grown in wastewater. companies operating at bench or pilot scale have not displayed an The separate operations that result in an algae biosolids product interest in using wastewater resources or integrating wastewater cannot be considered to be mutually exclusive. An upstream choice concerning nutrient source, reactor design, or reactor operation will 16 affect downstream harvesting and dewatering alternatives and

14 16

12 14

12 10 10 8 8 6 6

Number of Companies 4 4 Number of Companies

2 2

0 0 Bench Pilot Demo Full Bench Pilot Demo Full

Demonstrated Discussed or Mentioned Not Discussed Biofilm Reactors Hybrid Designs Closed Reactors Open Ponds

Fig. 1. Scale of algae production companies and involvement with wastewater treatment. Fig. 2. Scale of algae production companies and production approach. 700 L. Christenson, R. Sims / Biotechnology Advances 29 (2011) 686–702 constraints. Conversely, the choice of a particular harvesting or Chorus I, Bartram J. Toxic cyanobacteria in water: a guide to their public health consequences, monitoring, and management. Taylor & Francis; 1999. dewatering method will dictate what upstream conditions must be Correll DL. Role of phosphorus in the eutrophication of receiving waters: a review. J met. The use of a biofilm based system could more effectively and Environ Qual 1998;27:261–6. efficiently integrate production, harvesting, and dewatering opera- Danquah MK, Ang L, Uduman N, Moheimani N, Forde GM. Dewatering of microalgal culture for biodiesel production: exploring polymer flocculation and tangential tions; however, there is little information on the use of such a design flow filtration. 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