Bioresource Technology 102 (2011) 17–25

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The potential of sustainable algal biofuel production using wastewater resources Jon K. Pittman a,*, Andrew P. Dean a, Olumayowa Osundeko a,b a b

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK Sustainable Consumption Institute, University of Manchester, 188 Waterloo Place, Oxford Road, Manchester M13 9PL, UK

a r t i c l e

i n f o

Article history: Received 31 March 2010 Received in revised form 3 June 2010 Accepted 7 June 2010 Available online 1 July 2010 Keywords: Microalgae Biofuel Biomass Lipids Wastewater

a b s t r a c t The potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction

1.1. Algal biofuels

As the demand for energy continues to increase globally, fossil fuel usage will likewise continue to rise. There is still a plentiful supply of fossil fuels at reasonably low cost, although this is likely to change in the future, but more critically a rising use of fossil fuels is unlikely to be sustainable in the longer term principally due to the attributed increase in greenhouse gas (GHG) emissions from using these fuels and the environmental impact of these emissions on global warming (Hill et al., 2006). There is therefore significant interest in identifying alternative renewable sources of fuel that are potentially carbon neutral (Demirbas, 2009; Hill et al., 2006; Rittmann, 2008). The majority of the current commercially available biofuels are bioethanol derived from sugar cane or corn starch or biodiesel derived from oil crops including soybean and oilseed rape. Although biofuels have the potential to be environmentally beneficial compared to fossil fuels, there is some dispute as to whether these crop-based biofuels are economically competitive compared to fossil fuels. Furthermore, there is even more concern over the impact that the use of these crops for biofuels might have on food availability (Demirbas, 2009; Hill et al., 2006). Biofuels derived from the cultivation of algae have therefore been proposed as an alternative approach that does not impact on agriculture.

Algae, particularly green unicellular microalgae have been proposed for a long time as a potential renewable fuel source (Benemann et al., 1977; Oswald and Golueke, 1960). Microalgae have the potential to generate significant quantities of biomass and oil suitable for conversion to biodiesel. Microalgae have been estimated to have higher biomass productivity than plant crops in terms of land area required for cultivation, are predicted to have lower cost per yield, and have the potential to reduce GHG emissions through the replacement of fossil fuels (for reviews and further analysis see Benemann and Oswald, 1996; Brennan and Owende, 2010; Brune et al., 2009; Chisti, 2008; Dismukes et al., 2008; Huntley and Redalje, 2007; Rittmann, 2008; Schenk et al., 2008; Sheehan et al., 1998; Stephens et al., 2010). As with plant-derived feedstocks, algal feedstocks can be utilised directly or processed into liquid fuels and gas by a variety of biochemical conversion or thermochemical conversion processes (reviewed by Amin, 2009; Brennan and Owende, 2010; Demirbas, 2009; Rittmann, 2008). Dried algal biomass may be used to generate energy by direct combustion (Kadam, 2002) but this is probably the least attractive use for algal biomass. Thermochemical conversion methods include gasification, pyrolysis, hydrogenation and liquefaction of the algal biomass to yield gas- or oil-based biofuels (McKendry, 2002a,b; Miao and Wu, 2004). Biochemical conversion processes include fermentation and anaerobic digestion of the biomass to yield bioethanol or methane (McKendry, 2002a). In addition, hydrogen can be produced from algae by bio-

* Corresponding author. Tel.: +44 161 275 5235; fax: +44 161 275 5082. E-mail address: [email protected] (J.K. Pittman). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.035

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photolysis (Melis, 2002). Finally, lipids, principally triacylglycerol lipids can be separated and isolated from harvested microalgae and then converted to biodiesel by transesterification (Chisti, 2007; Hu et al., 2008; Miao and Wu, 2006). This latter process, the use of microalgae for biodiesel production has attracted a significant amount of interest. Research as part of the Aquatic Species Program funded by the US Department of Energy extensively analysed the oil production capabilities of microalgae and suggested that potential productivity of oil from microalgae may be significantly greater than oilseed crops such as soybean (Sheehan et al., 1998). This and subsequent research has focussed on identifying microalgae strains that are capable of synthesising significant quantities of lipids and in identifying cultivation conditions that will provide the greatest lipid productivities (Griffiths and Harrison, 2009; Hu et al., 2008). Many studies have focussed on identifying conditions that induce high accumulation of neutral lipids (particularly triacylglycerol) in the microalgae cells, like a nutrient stress such as nitrogen (N) or phosphorus (P) limitation (Converti et al., 2009; Dean et al., 2010; Li et al., 2008; Rodolfi et al., 2009). However, a major limitation of this approach is that despite inducing very high lipid yield, biomass productivity of the cells is often very low and so lipid productivity will not be high. Cultivation conditions that focus on providing high biomass productivity instead may ultimately be more beneficial and may be a more efficient means of increasing total lipid productivity (Griffiths and Harrison, 2009). Furthermore, with large quantities of algal biomass it may be more economically viable to generate energy via the production of the other types of biofuel. 1.2. The potential for sustainable biofuel production One of the attractions of microalgae as a biofuel feedstock is that they can be effectively grown in conditions which require minimal freshwater input unlike many plant-based biofuel crops, and utilise land which is otherwise non-productive to plant crops, thus making the process potentially sustainable with regard to preserving freshwater resources. For example, microalgae could be cultivated near the sea to utilise saline or brackish water. There has therefore been significant interest in the growth of microalgae for biofuels under saline conditions (e.g. Rodolfi et al., 2009; Takagi et al., 2006). However, another potentially sustainable growth medium for algal feedstock is wastewater. It has been appreciated for some years now that microalgae can be potentially utilised for low-cost and environmentally friendly wastewater treatment compared to other more commonly used treatment processes (de la Noue et al., 1992; Green et al., 1995; Oswald et al., 1957). The major problem with most wastewaters is the very high concentrations of nutrients, particularly total N and total P concentration as well as toxic metals, which require costly chemical-based treatments to remove them during wastewater treatment (Gasperi et al., 2008). Total N and P concentrations can be found at values of 10–100 mg L 1 in municipal wastewater and >1000 mg L 1 in agricultural effluent (de la Noue et al., 1992). The ability of microalgae to effectively grow in nutrient-rich environments and to efficiently accumulate nutrients and metals from the wastewater, make them an extremely attractive means for sustainable and low cost wastewater treatment (de-Bashan and Bashan, 2010; Hoffmann, 1998; Mallick, 2002). However, it has also long been proposed that wastewater-grown algae could be used for energy production (Benemann et al., 1977; Oswald and Golueke, 1960). There have been contrasting assessments as to the economic viability of algal biofuels. A number of studies have argued that biofuel production from algae, particularly biodiesel production is both economically and environmentally sustainable (Brune et al., 2009; Chisti, 2008; Huntley and Redalje, 2007; Stephens

Fig. 1. A flow-diagram showing how wastewater resources could be utilised for sustainable algal-based biofuel production.

et al., 2010), although there have been some sceptical views of the long term viability and economics of biofuels from algae (Reijnders, 2008; van Beilen, 2010; Walker, 2009). One frequent criticism is that the use of fossil fuels in the biofuel production process, in the construction of algal growth facilities, supply of nutrients for algal growth, harvesting of algae and biomass processing, is not often considered in the evaluation of algal biofuel viability and would in fact give rise to a net negative energy output. The use of wastewater resources may be a viable means to enhance the sustainability of algal biofuel production, both by providing a dual use process, an effective growth medium for algal cultivation, and freely available nutrient (particularly N and P) input (Fig. 1). This review will describe the ability of algae to grow in wastewater conditions, the uses of algae in wastewater processes, and the current research on the use of wastewater resources for potentially costeffective microalgal biofuel production. 2. Algae and wastewater Many species of microalgae are able to effectively grow in wastewater conditions through their ability to utilise abundant organic carbon and inorganic N and P in the wastewater. The use of microalgae in wastewater treatment has been long promoted (Oswald et al., 1957), however, chemical processing of waste or the generation of activated sludge is the conventional treatment method. Although the application of microalgae in the wastewater industry is still fairly limited, algae are used throughout the world for wastewater treatment albeit on a relatively minor scale. This is either through the use of conventional oxidation (stabilization) ponds or the more developed suspended algal pond systems such as high-rate algal ponds which are shallow raceway-type oxidation ponds with mechanical mixing, and have been shown to be highly effective for wastewater treatment (Green et al., 1995; Hoffmann, 1998). A major requirement of wastewater treatment is the need to remove high concentrations of nutrients in particular N and P, which otherwise can lead to risks of eutrophication if these nutrients accumulate in rivers and lakes. P is particularly difficult to remove from wastewater. For most commercial wastewater processing, P is precipitated from the effluent with the use of chemicals to form a solid insoluble fraction or is converted into an activated sludge by microbial activity (Hoffmann, 1998). However, the P recovered by these methods is not fully recyclable and the P precipitate is either buried in landfill or further treated to generate sludge fertiliser. Microalgae are efficient in removing N, P and toxic metals from

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wastewater (Ahluwalia and Goyal, 2007; Mallick, 2002) and therefore have potential to play an important remediation role particularly during the final (tertiary) treatment phase of wastewater. Indeed algae-based treatments have been found to be as efficient at removing P from wastewater as compared to chemical treatment (Hoffmann, 1998). The significant advantage of algal processes in wastewater treatment over the conventional chemical-based treatment methods is the potential cost saving and the lower level technology that is utilised, therefore making this approach more attractive to developing countries. For example, the significant O2 generation from photosynthetic microalgae will negate the need and therefore high operational cost of mechanical aeration of the treatment pond (Mallick, 2002). Oxygenation of treatment ponds is essential to allow efficient bioremediation of organic and inorganic compounds by heterotrophic aerobic bacteria (Munoz and Guieysse, 2006). Furthermore, an algal method of remediation is more environmentally amenable and sustainable as it does not generate additional pollutants such as sludge byproducts and provides an opportunity for efficient recycling of nutrients. For example, recovered N- and P-rich algal biomass can be used as low-cost fertiliser or as animal feed (Munoz and Guieysse, 2006; Wilkie and Mulbry, 2002). Most of the research on algal wastewater treatment has come from the analysis of laboratory-based small scale and pilot pondscale cultures, and from experimental high-rate algal ponds. A wide range of studies have analysed the growth of microalgae under a variety of wastewater conditions, mainly growth in municipal (urban) sewage wastewater and agricultural manure wastewater. These studies have principally been focussed on evaluating the potential of algae for removing N and P, and in some instances metals from wastewater. These initial experimental studies, particularly those that have also assessed variables for maximal algal biomass production and methods for harvesting algal biomass from wastewater, will be of significant benefit for the evaluation of wastewater-grown microalgae as a biofuel. 2.1. The efficiency of algal growth in wastewater The efficient growth of microalgae in wastewater depends on a variety of variables. As with any growth medium, critical variables are the pH and temperature of the growth medium, the concentration of essential nutrients, including N, P and organic carbon (and the ratios of these constituents), and the availability of light, O2 and CO2. For example, growth of microalgae in primary settled sewage water was shown to increase significantly under long photoperiod conditions and following addition of CO2, while increased temperature decreased algal biomass (Ip et al., 1982). A major difference between wastewater media and other growth media is the high concentration of nutrients in wastewater, such as N and P. Much of the N is often in the form of ammonia which at high concentration can inhibit algal growth (Ip et al., 1982; Konig et al., 1987; Wrigley and Toerien, 1990). The presence of toxins such cadmium or mercury, or organic chemicals is another critical factor of algal growth in wastewater. This will particularly be an issue with industrial-derived wastewaters. Biotic factors that may impact negatively on algal growth include pathogenic bacteria or predatory zooplankton. In addition, other microorganisms in the wastewater might out-compete the microalgae for essential nutrients. The starting density of microalgae in the wastewater is also likely to be a critical factor for the growth of the whole population (Lau et al., 1995). These variables will obviously differ depending on the wastewater type and from one wastewater treatment site to another. Furthermore, there will be variation in the ability of different algal species to tolerate a particular wastewater condition. Unicellular chlorophytic microalgae have been shown to be particularly toler-

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ant to many wastewater conditions and very efficient at accumulating nutrients from wastewater (Aslan and Kapdan, 2006; Gonzalez et al., 1997; Ruiz-Marin et al., 2010). Chlorella and Scenedesmus are usually predominant of the phytoplanktonic communities in oxidation ponds (Masseret et al., 2000) and in high-rate algal ponds (Canovas et al., 1996). Nevertheless there is variation in effectiveness between chlorophyte species. For example, Chlorella vulgaris was more effective than Chlorella kessleri at accumulating N and P from wastewater in one study (Travieso et al., 1992), while another study found that Scenedesmus obliquus grew better in municipal wastewater than C. vulgaris (Ruiz-Marin et al., 2010). In the rest of this section we will briefly review some of the studies of algal growth under four different wastewater conditions: municipal sewage wastewater, agricultural manure-based wastewater, industrial wastewater and artificial wastewater. 2.2. Algal growth in municipal sewage wastewater Conventional municipal sewage treatment consists of a primary treatment phase for the sedimentation of solid materials, a secondary treatment phase in which suspended and dissolved organic materials are removed, and a tertiary treatment phase in which final treatment of the water is performed prior to discharge into the environment. It is during this tertiary phase that the removal of many dissolved inorganic compounds including N and P takes place and it is the potential of microalgae in N and P removal during tertiary sewage treatment which has been assessed extensively. Some unicellular green microalgae species are particularly tolerant to sewage effluent conditions, most notably those of the Chlorella and Scenedesmus genus and so most studies have examined the growth of these species (e.g. Bhatnagar et al., 2010; Lau et al., 1995; Ruiz-Marin et al., 2010; Shi et al., 2007; Wang et al., 2010, in press). Microalgae have been shown to be very efficient at removing N and P from sewage-based wastewater either in a free-swimming suspension or in an immobilized form (see below). For example, various species of Chlorella and Scenedesmus can provide very high (>80%) and in many cases almost complete removal of ammonia, nitrate and total P from secondary treated wastewater (Martinez et al., 2000; Ruiz-Marin et al., 2010; Zhang et al., 2008), indicating the potential of microalgae for tertiary sewage treatment. Many of these experiments were performed under laboratory-based batch culture conditions with the microalgae showing high growth rates over the batch growth period. Ruiz-Marin et al. (2010) also compared growth of S. obliquus under semi-continuous culture conditions and found that initial growth over four cultivation cycles (every 35 h with fresh wastewater added at the start of each cycle) was much higher than in batch culture, possibly due to eventual nutrient depletion in the batch, but after four cycles of culture, growth and chlorophyll content of the cells decreased significantly, indicating a collapse of the culture. Studies have also shown microalgae to grow and efficiently remove nutrients from primary settled sewage wastewater. For example, C. vulgaris was demonstrated to remove over 90% of N content and 80% of P content from the primary treated sewage (Lau et al., 1995). This study compared the effect of varying the starting algal inoculum density with treatments ranging from a concentrated inoculum of 1  107 cells mL 1 to a low density inoculum of 5  105 cells mL 1 and found that growth rates were not significantly different between all treatments and apart from the lowest starting inoculum density, the total amounts of nutrients removed from all treatments were equivalent. This suggests that effective wastewater growth and nutrient removal is not significantly dependent on starting cell density. Two other recent analyses have assessed the growth of Chlorella sp. in raw sewage waste. Wang et al. (2010, in press) looked at the growth of Chlorella in pre-

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treated wastewater in addition to wastewater from three subsequent treatment phases. Many of the tested parameters including N and P removal, metal ion removal and growth rate were equivalent in wastewater prior to and after primary settling. Algal growth rate was significantly highest out of all four treatments in the fourth phase centrate wastewater which is the wastewater generated from the sludge centrifuge. This was likely due to the much higher concentration of N and P in this wastewater (131.5 mg L 1 total N and 201.5 mg L 1 total P) and although the N:P ratio was non-optimal compared to standard algal growth media, the cells were still able to grow well (Wang et al. (2010, in press)). A second recent study characterized a Chlorella species called Chlorella minutissima which was identified in wastewater treatment oxidation ponds in India (Bhatnagar et al., 2010). C. minutissima was able to grow well in high concentrations of raw sewage and dominate the subsequent pond stages in the oxidation pond system. Analysis has found that this species can grow heterotrophically in the dark, mixotrophically in the light utilising a variety of organic carbon substrates, over a wide pH range and in the presence of salt. Furthermore, it can utilise either ammonia or nitrate as an N source. The growth of this alga was shown to be highest under mixotrophic (photoheterotrophic) conditions with biomass productivity of 379 mg L 1 after 10 days growth compared to biomass of 73.03 mg L 1 under photoautotrophic conditions (Bhatnagar et al., 2010). This species could therefore be a good candidate for high biomass productivity in a wastewater high-rate pond system. All of these experiments also further demonstrate that chlorophytic microalgae such as Chlorella can grow well even in very raw wastewater conditions. 2.3. Algal growth in agricultural wastewater Compared to municipal domestic sewage-based wastewater, agricultural wastewater, which is often derived from manure, can be very high in N and P content (Wilkie and Mulbry, 2002). Despite these high nutrient concentrations, studies have demonstrated efficient growth of microalgae on agricultural waste, and as with municipal wastewater, microalgae are efficient at removing N and P from manure-based wastewater (An et al., 2003; Gonzalez et al., 1997; Wilkie and Mulbry, 2002). For example, the green alga Botryococcus braunii grew well in piggery wastewater containing 788 mg L 1 NO3 and removed 80% of the initial NO3 content (An et al., 2003). Studies of algal-mediated nutrient recovery from dairy manure have assessed the potential of benthic freshwater algae rather than planktonic (suspended) algae due to the potential higher nutrient uptake rates in some species of benthic algae (Mulbry et al., 2008; Mulbry and Wilkie, 2001; Wilkie and Mulbry, 2002). These species include Microspora willeana, Ulothrix sp. and Rhizoclonium hierglyphicum. Using a semi-continuous cultivation method where the benthic algae was grown in recycling wastewater with fresh manure added daily, algal growth rates and nutrient uptake were found to be high and equivalent to values from algae grown on municipal wastewater (Wilkie and Mulbry, 2002).

for large-scale generation of algal biomass. Furthermore, municipal and agricultural waste is likely to be more widely available and more uniform in characteristic than the variable constituents of different industrial wastewaters. However, one recent study which may suggest potential for some industrial wastewaters in providing resources for the generation of significant algal biomass came from the analysis of wastewater from carpet mill effluent (Chinnasamy et al., 2010). Carpet mill wastewater (and a small proportion of municipal wastewater) from the city of Dalton, GA, USA, makes up 100–115 million L of wastewater per day. The wastewater includes process chemicals and pigments used in the mills, plus a range of inorganic elements including low concentrations of metals, and relatively low concentrations of total P and N. This wastewater was shown to be low enough in toxins and had enough P and N to support algal growth, with two freshwater microalgae B. braunii and Chlorella saccharophila, and a marine alga Pleurochrysis carterae, able to grow particularly well on the untreated wastewater (Chinnasamy et al., 2010). With the very large amount of wastewater available from this industry a significant amount of biomass and potentially also biodiesel could be generated from this resource (see below). 2.5. Algal growth in artificial wastewater Some studies have examined algal growth and nutrient removal characteristics using artificial wastewater (Aslan and Kapdan, 2006; Lee and Lee, 2001; Voltolina et al., 1999). Utilisation of an artificial medium has benefits such as ease of use for initial laboratory-based experiments. It also allows for a simplified analysis of the major components in a wastewater medium without one needing to consider unknown variables such as biotic components. Most artificial wastewater media are composed of inorganic constituents including high concentrations of specific nutrients and will lack solid organic material and other potential toxins. Therefore there may be some drawbacks in using artificial wastewater to assess conditions in real wastewater. Direct comparisons of artificial wastewater with municipal wastewater have found that although nutrient removal rates are equivalent, microalgal growth rates are higher in artificial wastewater (Lau et al., 1995; Ruiz-Marin et al., 2010). This is likely due to increased toxicity of the real wastewaters, inhibitory or competitive effects of indigenous bacteria and protozoa, and by the different chemical composition of the wastewaters. 3. Use of wastewater for biofuel generation The ability of microalgae to grow well under certain wastewater conditions, as described above, has indicated the potential of these resources as suitable sustainable growth medium for biofuel feedstock. In this section we will briefly assess in more detail how effective wastewater resources are in providing significant algal biomass and whether this biomass can generate high amounts of lipids for biodiesel production.

2.4. Algal growth in industrial wastewater

3.1. Generation of oil for biodiesel from wastewater-grown algae

There is significant interest in the use of algae for remediation of industrial-derived wastewaters, predominantly for the removal of heavy metal pollutants (cadmium, chromium, zinc, etc.) and organic chemical toxins (hydrocarbons, biocides, and surfactants), rather than N and P (reviewed by Ahluwalia and Goyal, 2007; de-Bashan and Bashan, 2010; Mallick, 2002). Due to generally low N and P concentration and high toxin concentrations, algal growth rates are lower in many industrial wastewaters. Consequently, there is less potential for utilising industrial wastewaters

Microalgae can generate lipids sometimes at significant concentration. The type of lipids which accumulate, whether saturated fatty acids, polyunsaturated fatty acids, glycolipids or triacylglycerols, and the quantity of lipids produced (sometimes up to 80% of the cell dry weight [DW]) will depend on the microalgae species and the growth condition (Chisti, 2007; Griffiths and Harrison, 2009; Hu et al., 2008). Often the highest concentrations of lipids that are reported tend to be either from photo-bioreactor-grown cells or batch culture-grown cells in the laboratory, whereas high

Table 1 Comparison of biomass and lipid productivities in microalgae grown in various wastewater conditions. Microalgae species

Biomass (DW) productivity (mg L 1 day 1)

Lipid content (% DW)

Lipid productivity (mg L 1 day 1)

References

Municipal (primary treated) Municipal (centrate) Municipal (secondary treated) Municipal (secondary treated) Municipal (primary treated + CO2) Agricultural (piggery manure with high NO3-N) Agricultural (dairy manure with polystyrene foam support) Agricultural (fermented swine urine) Agricultural (anaerobically digested dairy manure)

nd Chlamydomonas reinhardtii (biocoil-grown) Scenedesmus obliquus Botryococcus braunii Mix of Chlorella sp., Micractinium sp., Actinastrum sp. B. braunii Chlorella sp. Scenedesmus sp. Mix of Microspora willeana, Ulothrix zonata, Ulothrix aequalis, Rhizoclonium hieroglyphicum, Oedogonium sp. R. hieroglyphicum R. hieroglyphicum

25a 2000 26b 345.6c 270.7d 700e 2.6 g m 6f 5.5 g m

nd 25.25 31.4i 17.85 9 nd 9i 0.9i nd

nd 505 8i 62 24.4 69 230img m 0.54i nd

Ip et al. (1982) Kong et al. (2010) Martinez et al. (2000) Orpez et al. (2009) Woertz et al. (2009) An et al. (2003) Johnson and Wen (2010) Kim et al. (2007) Wilkie and Mulbry (2002)

10.7 g m 17.9 g m

0.7i 1.2i

72i mg m 2 day 1 210i mg m 2 day 1

Mulbry et al. (2008) Mulbry et al. (2008)

Chlorella sp.

81.4g

13.6i

11i

29

17

Wang et al. (2010, in press) Woertz et al. (2009)

13.20 18.10 15.20 12.00 12.8

4.5 4.2 4.3 4.0 16.2

Chinnasamy et al. (2010) Chinnasamy et al. (2010) Chinnasamy et al. (2010) Chinnasamy et al. (2010) Voltolina et al. (1999)

Agricultural (swine effluent, maximum manure loading rate) Agricultural (dairy effluent + CO2, maximum manure loading rate) Agricultural (digested dairy manure, 20 dilution) Agricultural (dairy wastewater, 25% dilution) Industrial (carpet mill, Industrial (carpet mill, Industrial (carpet mill, Industrial (carpet mill, Artificial wastewater

untreated) untreated) untreated) untreated)

Mix of Chlorella sp., Micractinium sp., Actinastrum sp. B. braunii Chlorella saccharophila Dunaliella tertiolecta Pleurochrysis carterae Scenedesmus sp.

nd – not determined; DW – dry weight. a Estimated from biomass value of 1000 mg L 1 after 40 days. b Estimated from biomass value of 1.1 mg L 1 h 1. c Estimated from biomass value of 14.4 mg L 1 h 1. d Estimated from biomass value of 812 mg L 1 after 3 days. e Estimated from biomass value of 7 g L 1 after 10 days. f Estimated from biomass value of 197 mg L 1 after 31 days. g Estimated from biomass value of 1.71 g L 1 after 21 days. h Estimated from lipid productivity and lipid content value. i Fatty acid content and productivity determined rather than total lipid.

59

h

34 23 28 33 126.54

2

day

1

2

day

1

2 2

day day

1 1

2

day

1

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Wastewater type

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concentrations of lipid tend not to be observed in open pondgrown microalgae (Griffiths and Harrison, 2009). Furthermore, these very high concentrations of cell lipid tend to be coupled with low biomass particularly when induced by environmental stress such as N or P limitation (Dean et al., 2010; Rodolfi et al., 2009), therefore the determination of lipid productivities from high biomass culture conditions such as wastewater-grown microalgae are of particular interest. A number of recent laboratory-based studies where microalgae have been grown either in small batch cultures, small semi-continuous cultures or bioreactors have reported reasonable lipid accumulation in wastewater-grown microalgae, ranging from low (95%), although a major disadvantage is that it is an energy intensive process. A further method of harvesting is filtration. Filtration using pressure of vacuum, together with the use of filter aids such as diatomaceous earth or cellulose are suitable for the recovery of larger algae (>70 lm) but not for smaller microalgae such as Scenedesmus and Chlorella (Brennan and Owende, 2010). For small cells, membrane microfiltration and ultrafiltration are alternative methods, however, the need to frequently replace membranes and the cost of pumping makes this an expensive process. In general, centrifugation remains the most reliable and preferred method for algal harvesting, and is only slightly more expensive than alternative methods (Olaizola, 2003). An alternative method for harvesting microalgae is immobilization of the microalgae prior to and during cultivation so that the biomass can be easily retrieved at the end of the growth period. This harvesting method is particularly useful for water treatment applications, and various methods for this have been developed and evaluated for their efficiency of pollutant removal from wastewater (reviewed by de-Bashan and Bashan, 2010; Mallick, 2002). Artificial attachment or polymer encapsulation of microalgae, such as in alginate, are the most frequently used immobilization approaches. An effective method must maintain live cells for as long as possible and allow high flow of the waste medium into the polymer-algae matrix to allow high nutrient accumulation by the cells. Alginate-immobilized microalgae have been shown to be as effective as free-swimming microalgae in accumulating N and P from municipal wastewater. For example, C. vulgaris immobilized in alginate pellets placed in a fluidized bed column could effectively remove approximately 80% of the ammonia content and 70% of the total P content in a sewage culture (Travieso et al., 1992). Similarly, Scenedesmus immobilized in alginate sheets could effectively remove ammonia and orthophosphate from secondary treated effluent (Zhang et al., 2008). Algal biomass can be recovered from an immobilized matrix and this may be suitable feedstock for downstream thermochemical processing such as pyrolysis or for the generation of bioethanol by fermentation. However, it is unclear whether immobilized algae could be used for oil production and whether lipids could be efficiently removed from an algal– polymer matrix. Following harvesting, the algae have to go through a number of further processes, however the issues of downstream microalgae

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processing, conversion and oil extraction, and the need for improvements in this part of the algal biofuel production process have been discussed extensively in other articles and will not be reviewed here (Amin, 2009; Brennan and Owende, 2010). 4.2. The need for life cycle analysis of wastewater-derived algal biofuel A critical determinant as to the true potential of algal biofuel production using wastewater resources is whether the process will provide a positive energy output which will also determine the economic viability of the process. In addition there will be a need to determine whether the process is truly sustainable in terms of the utilisation of natural resources and whether the process is truly carbon neutral. Results from recent life cycle energy assessments of biofuel feedstocks including microalgae and in comparison to other bioenergy feedstocks have provided mixed conclusions (Clarens et al., 2010; Lardon et al., 2009). Lardon et al. (2009) and Clarens et al. (2010) have both performed life cycle assessments of biofuel production from microalgae in comparison to other plantbased biofuels and fossil fuel. Both assessments concluded that based on current technology parameters, algal-based biofuels had a positive energy balance but performed relatively poorly compared to other biofuels. Clarens et al. (2010) suggested that plant-based biofuels have lower energy use, GHG emissions and water use than algae biofuels. It was concluded that a major factor in the poor environmental impact of algal biofuels as modelled in this assessment, was the demand for CO2 and fertiliser as a nutrient source. Wastewater use could offset these nutrient and CO2 demands. As well as being high in nutrients, many wastewaters, particularly municipal and agricultural wastewaters, have significant aerobic bacterial populations which will generate CO2 through respiration and which can be utilised by the microalgae (Munoz and Guieysse, 2006). Clarens et al. (2010) further modelled algalbased biofuel production when coupled to three types of municipal and agricultural waste as sources for N and P. These models assumed the addition of wastewater-derived nutrient to algae in a freshwater-based pond. All three models significantly improved the life cycle burden on the algal biofuel production and when source-separated urine was modelled as the wastewater resource, the algal-based process was shown to be more environmentally beneficial than terrestrial plant biofuel crops. Further life cycle analysis is needed to assess the alternative wastewater strategies reviewed here, which include using wastewater resources not only as an added nutrient supply but also as the pond medium, and potentially as the driver for the energy input and additional CO2 input (Fig. 1). 4.3. The need to demonstrate high biomass and lipid productivity at pond scale As described above, microalgae biomass can be processed and converted to biofuel by a variety of methods, however, lipid extraction from microalgae and the use of microalgae as an oil source for biodiesel production is likely to be one of the most attractive options, particularly if the remaining residual algal biomass is utilised for biogas production (Brune et al., 2009). Although most microalgae have relatively low total lipid content per cell under wastewater conditions (Table 1), the potentially high biomass productivity will translate to significant total lipid productivity. Some of the initial studies of wastewater-grown microalgae have reported very significant biomass yield (e.g. Kong et al., 2010) and suggest much promise but future work must demonstrate similar yields on a larger scale and in open pond conditions. Most of the studies to date have analysed microalgae biomass and lipid production under laboratory conditions and following microalgae cultivation in batch culture flasks or bioreactors rather than in ponds. There is also a

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need to demonstrate that these biomass yields can be maintained over long cultivation periods. There is also the possibility that novel technologies such as genetic manipulation of microalgae, which is being used to attempt to improve algal lipid content (e.g. Wang et al., 2009), could likewise be used to improve algal growth and/or lipid production under wastewater conditions. An alternative method to improve the lipid productivity of rapidly growing microalgae is the recently described photosynthesis– fermentation model (PFM) for microalgal cultivation (Xiong et al., 2010). Autotrophically grown cells (photosynthesis growth) grow to high cell densities but usually have relatively low amounts of lipid per cell. Previous studies have demonstrated that heterotrophic fermentation of microalgae species which are capable of growing heterotrophically, such as Chlorella protothecoides, can provide high biomass production and high lipid content (Miao and Wu, 2004, 2006). The study of Xiong et al. (2010) evaluated the integrated strategy of algal cultivation whereby an initial autotrophic growth phase in which high biomass was generated, was then followed by a heterotrophic fermentation phase to maximise cell density and lipid accumulation. Interestingly, lipid yield of C. protothecoides was higher by nearly 70% following this PFM mode of cultivation compared to heterotrophic fermentation lacking any autotrophic growth, indicating a higher conversion efficiency of sugar to oil during PFM growth. This new approach has the potential to further enhance lipid productivity and therefore increase the efficiency of microalgal biodiesel production. Furthermore, such a strategy may be an ideal means to improve the lipid productivity of microalgae cultivated on organic carbon-rich wastewater resources. 5. Conclusions Based on current technologies algal cultivation for biofuel production alone is unlikely to be economically viable or provide a positive energy return. Dual-use microalgae cultivation for wastewater treatment coupled with biofuel generation is therefore an attractive option in terms of reducing the energy cost, GHG emissions, and the nutrient (fertiliser) and freshwater resource costs of biofuel generation from microalgae. The high biomass productivity of wastewater-grown microalgae suggests that this cultivation method offers real potential as a viable means for biofuel generation and is likely to be one of many approaches used for the production of sustainable and renewable energy. Acknowledgements O.O. is grateful to the Sustainable Consumption Institute Doctoral Training Centre for providing a graduate studentship. J.K.P. is grateful to The Leverhulme Trust and The Carbon Trust for financial support. References Ahluwalia, S.S., Goyal, D., 2007. Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresour. Technol. 98, 2243–2257. Amin, S., 2009. Review on biofuel oil and gas production processes from microalgae. Energy Convers. Manag. 50, 1834–1840. An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii. J. Appl. Phycol. 15, 185–191. Aslan, S., Kapdan, I.K., 2006. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecol. Eng. 28, 64–70. Benemann, J.R., Oswald, W.J., 1996. Systems and Economic Analysis o Microalgae Ponds for Conversion of CO2 to Biomass. Department of Energy Pittsburgh Energy Technology Center Final Report, Grant No. DE-FG22-93PC93204. Benemann, J.R., Weissman, J.C., Koopman, B.L., Oswald, W.J., 1977. Energy production by microbial photosynthesis. Nature 268, 19–23. Bhatnagar, A., Bhatnagar, M., Chinnasamy, S., Das, K., 2010. Chlorella minutissima – a promising fuel alga for cultivation in municipal wastewaters. Appl. Biochem. Biotechnol. 161, 523–536.

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