Bioresource Technology 102 (2011) 17–25

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

The potential of sustainable algal biofuel production using wastewater resources

Jon K. Pittman a,*, Andrew P. Dean a, Olumayowa Osundeko a,b
a
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK
b
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 a b s t r a c t

Article history: The potential of microalgae as a source of renewable energy has received considerable interest, but if mic-
Received 31 March 2010 roalgal biofuel production is to be economically viable and sustainable, further optimization of mass cul-
Received in revised form 3 June 2010 ture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities
Accepted 7 June 2010
potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there
Available online 1 July 2010
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
Keywords:
and limitations of using wastewaters as resources for cost-effective microalgal biofuel production.
Microalgae
Biofuel
Ó 2010 Elsevier Ltd. All rights reserved.
Biomass
Lipids
Wastewater

1. Introduction 1.1. Algal biofuels

As the demand for energy continues to increase globally, fossil Algae, particularly green unicellular microalgae have been pro-
fuel usage will likewise continue to rise. There is still a plentiful posed for a long time as a potential renewable fuel source (Bene-
supply of fossil fuels at reasonably low cost, although this is likely mann et al., 1977; Oswald and Golueke, 1960). Microalgae have
to change in the future, but more critically a rising use of fossil the potential to generate significant quantities of biomass and oil
fuels is unlikely to be sustainable in the longer term principally suitable for conversion to biodiesel. Microalgae have been esti-
due to the attributed increase in greenhouse gas (GHG) emissions mated to have higher biomass productivity than plant crops in
from using these fuels and the environmental impact of these terms of land area required for cultivation, are predicted to have
emissions on global warming (Hill et al., 2006). There is therefore lower cost per yield, and have the potential to reduce GHG emis-
significant interest in identifying alternative renewable sources sions through the replacement of fossil fuels (for reviews and fur-
of fuel that are potentially carbon neutral (Demirbas, 2009; Hill ther analysis see Benemann and Oswald, 1996; Brennan and
et al., 2006; Rittmann, 2008). The majority of the current commer- Owende, 2010; Brune et al., 2009; Chisti, 2008; Dismukes et al.,
cially available biofuels are bioethanol derived from sugar cane or 2008; Huntley and Redalje, 2007; Rittmann, 2008; Schenk et al.,
corn starch or biodiesel derived from oil crops including soybean 2008; Sheehan et al., 1998; Stephens et al., 2010).
and oilseed rape. Although biofuels have the potential to be envi- As with plant-derived feedstocks, algal feedstocks can be uti-
ronmentally beneficial compared to fossil fuels, there is some dis- lised directly or processed into liquid fuels and gas by a variety
pute as to whether these crop-based biofuels are economically of biochemical conversion or thermochemical conversion pro-
competitive compared to fossil fuels. Furthermore, there is even cesses (reviewed by Amin, 2009; Brennan and Owende, 2010;
more concern over the impact that the use of these crops for bio- Demirbas, 2009; Rittmann, 2008). Dried algal biomass may be used
fuels might have on food availability (Demirbas, 2009; Hill et al., to generate energy by direct combustion (Kadam, 2002) but this is
2006). Biofuels derived from the cultivation of algae have therefore probably the least attractive use for algal biomass. Thermochemi-
been proposed as an alternative approach that does not impact on cal conversion methods include gasification, pyrolysis, hydrogena-
agriculture. tion 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 diges-
tion of the biomass to yield bioethanol or methane (McKendry,
* Corresponding author. Tel.: +44 161 275 5235; fax: +44 161 275 5082. 2002a). In addition, hydrogen can be produced from algae by bio-
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
18 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25

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 produc-
tion 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 culti-
vation 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
Fig. 1. A flow-diagram showing how wastewater resources could be utilised for
cells, like a nutrient stress such as nitrogen (N) or phosphorus (P)
sustainable algal-based biofuel production.
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 et al., 2010), although there have been some sceptical views of
high. Cultivation conditions that focus on providing high biomass the long term viability and economics of biofuels from algae (Reijn-
productivity instead may ultimately be more beneficial and may ders, 2008; van Beilen, 2010; Walker, 2009). One frequent criticism
be a more efficient means of increasing total lipid productivity is that the use of fossil fuels in the biofuel production process, in
(Griffiths and Harrison, 2009). Furthermore, with large quantities the construction of algal growth facilities, supply of nutrients for
of algal biomass it may be more economically viable to generate algal growth, harvesting of algae and biomass processing, is not of-
energy via the production of the other types of biofuel. ten considered in the evaluation of algal biofuel viability and
would in fact give rise to a net negative energy output. The use
1.2. The potential for sustainable biofuel production of wastewater resources may be a viable means to enhance the
sustainability of algal biofuel production, both by providing a dual
One of the attractions of microalgae as a biofuel feedstock is use process, an effective growth medium for algal cultivation, and
that they can be effectively grown in conditions which require freely available nutrient (particularly N and P) input (Fig. 1). This
minimal freshwater input unlike many plant-based biofuel crops, review will describe the ability of algae to grow in wastewater con-
and utilise land which is otherwise non-productive to plant crops, ditions, the uses of algae in wastewater processes, and the current
thus making the process potentially sustainable with regard to pre- research on the use of wastewater resources for potentially cost-
serving freshwater resources. For example, microalgae could be effective microalgal biofuel production.
cultivated near the sea to utilise saline or brackish water. There
has therefore been significant interest in the growth of microalgae 2. Algae and wastewater
for biofuels under saline conditions (e.g. Rodolfi et al., 2009; Takagi
et al., 2006). However, another potentially sustainable growth Many species of microalgae are able to effectively grow in
medium for algal feedstock is wastewater. wastewater conditions through their ability to utilise abundant or-
It has been appreciated for some years now that microalgae can ganic carbon and inorganic N and P in the wastewater. The use of
be potentially utilised for low-cost and environmentally friendly microalgae in wastewater treatment has been long promoted (Os-
wastewater treatment compared to other more commonly used wald et al., 1957), however, chemical processing of waste or the
treatment processes (de la Noue et al., 1992; Green et al., 1995; Os- generation of activated sludge is the conventional treatment meth-
wald et al., 1957). The major problem with most wastewaters is od. Although the application of microalgae in the wastewater
the very high concentrations of nutrients, particularly total N and industry is still fairly limited, algae are used throughout the world
total P concentration as well as toxic metals, which require costly for wastewater treatment albeit on a relatively minor scale. This is
chemical-based treatments to remove them during wastewater either through the use of conventional oxidation (stabilization)
treatment (Gasperi et al., 2008). Total N and P concentrations can ponds or the more developed suspended algal pond systems such
be found at values of 10–100 mg L 1 in municipal wastewater as high-rate algal ponds which are shallow raceway-type oxidation
and >1000 mg L 1 in agricultural effluent (de la Noue et al., ponds with mechanical mixing, and have been shown to be highly
1992). The ability of microalgae to effectively grow in nutrient-rich effective for wastewater treatment (Green et al., 1995; Hoffmann,
environments and to efficiently accumulate nutrients and metals 1998).
from the wastewater, make them an extremely attractive means A major requirement of wastewater treatment is the need to re-
for sustainable and low cost wastewater treatment (de-Bashan move high concentrations of nutrients in particular N and P, which
and Bashan, 2010; Hoffmann, 1998; Mallick, 2002). However, it otherwise can lead to risks of eutrophication if these nutrients
has also long been proposed that wastewater-grown algae could accumulate in rivers and lakes. P is particularly difficult to remove
be used for energy production (Benemann et al., 1977; Oswald from wastewater. For most commercial wastewater processing, P
and Golueke, 1960). is precipitated from the effluent with the use of chemicals to form
There have been contrasting assessments as to the economic a solid insoluble fraction or is converted into an activated sludge by
viability of algal biofuels. A number of studies have argued that microbial activity (Hoffmann, 1998). However, the P recovered by
biofuel production from algae, particularly biodiesel production these methods is not fully recyclable and the P precipitate is either
is both economically and environmentally sustainable (Brune buried in landfill or further treated to generate sludge fertiliser.
et al., 2009; Chisti, 2008; Huntley and Redalje, 2007; Stephens Microalgae are efficient in removing N, P and toxic metals from
 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25 19

wastewater (Ahluwalia and Goyal, 2007; Mallick, 2002) and there- ant to many wastewater conditions and very efficient at accumu-
fore have potential to play an important remediation role particu- lating nutrients from wastewater (Aslan and Kapdan, 2006;
larly during the final (tertiary) treatment phase of wastewater. Gonzalez et al., 1997; Ruiz-Marin et al., 2010). Chlorella and Scene-
Indeed algae-based treatments have been found to be as efficient desmus are usually predominant of the phytoplanktonic communi-
at removing P from wastewater as compared to chemical treat- ties in oxidation ponds (Masseret et al., 2000) and in high-rate algal
ment (Hoffmann, 1998). The significant advantage of algal ponds (Canovas et al., 1996). Nevertheless there is variation in
processes in wastewater treatment over the conventional chemi- effectiveness between chlorophyte species. For example, Chlorella
cal-based treatment methods is the potential cost saving and the vulgaris was more effective than Chlorella kessleri at accumulating
lower level technology that is utilised, therefore making this ap- N and P from wastewater in one study (Travieso et al., 1992), while
proach more attractive to developing countries. For example, the another study found that Scenedesmus obliquus grew better in
significant O2 generation from photosynthetic microalgae will municipal wastewater than C. vulgaris (Ruiz-Marin et al., 2010).
negate the need and therefore high operational cost of mechanical In the rest of this section we will briefly review some of the studies
aeration of the treatment pond (Mallick, 2002). Oxygenation of of algal growth under four different wastewater conditions: muni-
treatment ponds is essential to allow efficient bioremediation of cipal sewage wastewater, agricultural manure-based wastewater,
organic and inorganic compounds by heterotrophic aerobic bacte- industrial wastewater and artificial wastewater.
ria (Munoz and Guieysse, 2006). Furthermore, an algal method of
remediation is more environmentally amenable and sustainable 2.2. Algal growth in municipal sewage wastewater
as it does not generate additional pollutants such as sludge by-
products and provides an opportunity for efficient recycling of Conventional municipal sewage treatment consists of a primary
nutrients. For example, recovered N- and P-rich algal biomass treatment phase for the sedimentation of solid materials, a second-
can be used as low-cost fertiliser or as animal feed (Munoz and ary treatment phase in which suspended and dissolved organic
Guieysse, 2006; Wilkie and Mulbry, 2002). materials are removed, and a tertiary treatment phase in which fi-
Most of the research on algal wastewater treatment has come nal treatment of the water is performed prior to discharge into the
from the analysis of laboratory-based small scale and pilot pond- environment. It is during this tertiary phase that the removal of
scale cultures, and from experimental high-rate algal ponds. A many dissolved inorganic compounds including N and P takes
wide range of studies have analysed the growth of microalgae place and it is the potential of microalgae in N and P removal dur-
under a variety of wastewater conditions, mainly growth in ing tertiary sewage treatment which has been assessed exten-
municipal (urban) sewage wastewater and agricultural manure sively. Some unicellular green microalgae species are particularly
wastewater. These studies have principally been focussed on eval- tolerant to sewage effluent conditions, most notably those of the
uating the potential of algae for removing N and P, and in some in- Chlorella and Scenedesmus genus and so most studies have exam-
stances metals from wastewater. These initial experimental ined the growth of these species (e.g. Bhatnagar et al., 2010; Lau
studies, particularly those that have also assessed variables for et al., 1995; Ruiz-Marin et al., 2010; Shi et al., 2007; Wang et al.,
maximal algal biomass production and methods for harvesting 2010, in press).
algal biomass from wastewater, will be of significant benefit for Microalgae have been shown to be very efficient at removing N
the evaluation of wastewater-grown microalgae as a biofuel. and P from sewage-based wastewater either in a free-swimming
suspension or in an immobilized form (see below). For example,
2.1. The efficiency of algal growth in wastewater various species of Chlorella and Scenedesmus can provide very high
(>80%) and in many cases almost complete removal of ammonia,
The efficient growth of microalgae in wastewater depends on a nitrate and total P from secondary treated wastewater (Martinez
variety of variables. As with any growth medium, critical variables et al., 2000; Ruiz-Marin et al., 2010; Zhang et al., 2008), indicating
are the pH and temperature of the growth medium, the concentra- the potential of microalgae for tertiary sewage treatment. Many of
tion of essential nutrients, including N, P and organic carbon (and these experiments were performed under laboratory-based batch
the ratios of these constituents), and the availability of light, O2 and culture conditions with the microalgae showing high growth rates
CO2. For example, growth of microalgae in primary settled sewage over the batch growth period. Ruiz-Marin et al. (2010) also com-
water was shown to increase significantly under long photoperiod pared growth of S. obliquus under semi-continuous culture condi-
conditions and following addition of CO2, while increased temper- tions and found that initial growth over four cultivation cycles
ature decreased algal biomass (Ip et al., 1982). A major difference (every 35 h with fresh wastewater added at the start of each cycle)
between wastewater media and other growth media is the high was much higher than in batch culture, possibly due to eventual
concentration of nutrients in wastewater, such as N and P. Much nutrient depletion in the batch, but after four cycles of culture,
of the N is often in the form of ammonia which at high concentra- growth and chlorophyll content of the cells decreased significantly,
tion can inhibit algal growth (Ip et al., 1982; Konig et al., 1987; indicating a collapse of the culture.
Wrigley and Toerien, 1990). The presence of toxins such cadmium Studies have also shown microalgae to grow and efficiently re-
or mercury, or organic chemicals is another critical factor of algal move nutrients from primary settled sewage wastewater. For
growth in wastewater. This will particularly be an issue with example, C. vulgaris was demonstrated to remove over 90% of N
industrial-derived wastewaters. Biotic factors that may impact content and 80% of P content from the primary treated sewage
negatively on algal growth include pathogenic bacteria or preda- (Lau et al., 1995). This study compared the effect of varying the
tory zooplankton. In addition, other microorganisms in the waste- starting algal inoculum density with treatments ranging from a
water might out-compete the microalgae for essential nutrients. concentrated inoculum of 1  107 cells mL 1 to a low density inoc-
The starting density of microalgae in the wastewater is also likely ulum of 5  105 cells mL 1 and found that growth rates were not
to be a critical factor for the growth of the whole population (Lau significantly different between all treatments and apart from the
et al., 1995). lowest starting inoculum density, the total amounts of nutrients
These variables will obviously differ depending on the waste- removed from all treatments were equivalent. This suggests that
water type and from one wastewater treatment site to another. effective wastewater growth and nutrient removal is not signifi-
Furthermore, there will be variation in the ability of different algal cantly dependent on starting cell density. Two other recent analy-
species to tolerate a particular wastewater condition. Unicellular ses have assessed the growth of Chlorella sp. in raw sewage waste.
chlorophytic microalgae have been shown to be particularly toler- Wang et al. (2010, in press) looked at the growth of Chlorella in pre-
20 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25

treated wastewater in addition to wastewater from three subse- for large-scale generation of algal biomass. Furthermore, municipal
quent treatment phases. Many of the tested parameters including and agricultural waste is likely to be more widely available and
N and P removal, metal ion removal and growth rate were equiva- more uniform in characteristic than the variable constituents of
lent in wastewater prior to and after primary settling. Algal growth different industrial wastewaters. However, one recent study which
rate was significantly highest out of all four treatments in the may suggest potential for some industrial wastewaters in provid-
fourth phase centrate wastewater which is the wastewater gener- ing resources for the generation of significant algal biomass came
ated from the sludge centrifuge. This was likely due to the much from the analysis of wastewater from carpet mill effluent (Chin-
higher concentration of N and P in this wastewater (131.5 mg L 1 nasamy et al., 2010). Carpet mill wastewater (and a small propor-
total N and 201.5 mg L 1 total P) and although the N:P ratio was tion of municipal wastewater) from the city of Dalton, GA, USA,
non-optimal compared to standard algal growth media, the cells makes up 100–115 million L of wastewater per day. The wastewa-
were still able to grow well (Wang et al. (2010, in press)). ter includes process chemicals and pigments used in the mills, plus
A second recent study characterized a Chlorella species called a range of inorganic elements including low concentrations of met-
Chlorella minutissima which was identified in wastewater treat- als, and relatively low concentrations of total P and N. This waste-
ment oxidation ponds in India (Bhatnagar et al., 2010). C. minutiss- water was shown to be low enough in toxins and had enough P and
ima was able to grow well in high concentrations of raw sewage N to support algal growth, with two freshwater microalgae B. brau-
and dominate the subsequent pond stages in the oxidation pond nii and Chlorella saccharophila, and a marine alga Pleurochrysis car-
system. Analysis has found that this species can grow heterotro- terae, able to grow particularly well on the untreated wastewater
phically in the dark, mixotrophically in the light utilising a variety (Chinnasamy et al., 2010). With the very large amount of wastewa-
of organic carbon substrates, over a wide pH range and in the pres- ter available from this industry a significant amount of biomass
ence of salt. Furthermore, it can utilise either ammonia or nitrate and potentially also biodiesel could be generated from this re-
as an N source. The growth of this alga was shown to be highest source (see below).
under mixotrophic (photoheterotrophic) conditions with biomass
productivity of 379 mg L 1 after 10 days growth compared to bio- 2.5. Algal growth in artificial wastewater
mass of 73.03 mg L 1 under photoautotrophic conditions (Bhatna-
gar et al., 2010). This species could therefore be a good candidate Some studies have examined algal growth and nutrient removal
for high biomass productivity in a wastewater high-rate pond sys- characteristics using artificial wastewater (Aslan and Kapdan,
tem. All of these experiments also further demonstrate that chloro- 2006; Lee and Lee, 2001; Voltolina et al., 1999). Utilisation of an
phytic microalgae such as Chlorella can grow well even in very raw artificial medium has benefits such as ease of use for initial labora-
wastewater conditions. tory-based experiments. It also allows for a simplified analysis of
the major components in a wastewater medium without one need-
2.3. Algal growth in agricultural wastewater ing to consider unknown variables such as biotic components.
Most artificial wastewater media are composed of inorganic con-
Compared to municipal domestic sewage-based wastewater, stituents including high concentrations of specific nutrients and
agricultural wastewater, which is often derived from manure, can will lack solid organic material and other potential toxins. There-
be very high in N and P content (Wilkie and Mulbry, 2002). Despite fore there may be some drawbacks in using artificial wastewater
these high nutrient concentrations, studies have demonstrated to assess conditions in real wastewater. Direct comparisons of arti-
efficient growth of microalgae on agricultural waste, and as with ficial wastewater with municipal wastewater have found that
municipal wastewater, microalgae are efficient at removing N although nutrient removal rates are equivalent, microalgal growth
and P from manure-based wastewater (An et al., 2003; Gonzalez rates are higher in artificial wastewater (Lau et al., 1995; Ruiz-Mar-
et al., 1997; Wilkie and Mulbry, 2002). For example, the green alga in et al., 2010). This is likely due to increased toxicity of the real
Botryococcus braunii grew well in piggery wastewater containing wastewaters, inhibitory or competitive effects of indigenous bacte-
788 mg L 1 NO3 and removed 80% of the initial NO3 content (An ria and protozoa, and by the different chemical composition of the
et al., 2003). wastewaters.
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 3. Use of wastewater for biofuel generation
uptake rates in some species of benthic algae (Mulbry et al.,
2008; Mulbry and Wilkie, 2001; Wilkie and Mulbry, 2002). These The ability of microalgae to grow well under certain wastewater
species include Microspora willeana, Ulothrix sp. and Rhizoclonium conditions, as described above, has indicated the potential of these
hierglyphicum. Using a semi-continuous cultivation method where resources as suitable sustainable growth medium for biofuel feed-
the benthic algae was grown in recycling wastewater with fresh stock. In this section we will briefly assess in more detail how
manure added daily, algal growth rates and nutrient uptake were effective wastewater resources are in providing significant algal
found to be high and equivalent to values from algae grown on mu- biomass and whether this biomass can generate high amounts of
nicipal wastewater (Wilkie and Mulbry, 2002). 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 Microalgae can generate lipids sometimes at significant concen-
of industrial-derived wastewaters, predominantly for the removal tration. The type of lipids which accumulate, whether saturated
of heavy metal pollutants (cadmium, chromium, zinc, etc.) and or- fatty acids, polyunsaturated fatty acids, glycolipids or triacylglyce-
ganic chemical toxins (hydrocarbons, biocides, and surfactants), rols, and the quantity of lipids produced (sometimes up to 80% of
rather than N and P (reviewed by Ahluwalia and Goyal, 2007; the cell dry weight [DW]) will depend on the microalgae species
de-Bashan and Bashan, 2010; Mallick, 2002). Due to generally and the growth condition (Chisti, 2007; Griffiths and Harrison,
low N and P concentration and high toxin concentrations, algal 2009; Hu et al., 2008). Often the highest concentrations of lipids
growth rates are lower in many industrial wastewaters. Conse- that are reported tend to be either from photo-bioreactor-grown
quently, there is less potential for utilising industrial wastewaters 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.

Wastewater type Microalgae species Biomass (DW) Lipid Lipid productivity References
productivity content (mg L 1 day 1)
(mg L 1 day 1) (% DW)
Municipal (primary treated) nd 25a nd nd Ip et al. (1982)
Municipal (centrate) Chlamydomonas reinhardtii (biocoil-grown) 2000 25.25 505 Kong et al. (2010)
Municipal (secondary treated) Scenedesmus obliquus 26b 31.4i 8i Martinez et al. (2000)
Municipal (secondary treated) Botryococcus braunii 345.6c 17.85 62 Orpez et al. (2009)
Municipal (primary treated + CO2) Mix of Chlorella sp., Micractinium sp., Actinastrum sp. 270.7d 9 24.4 Woertz et al. (2009)
Agricultural (piggery manure with high NO3-N) B. braunii 700e nd 69 An et al. (2003)
2 1
Agricultural (dairy manure with polystyrene foam support) Chlorella sp. 2.6 g m day 9i 230img m 2
day 1
Johnson and Wen (2010)
Agricultural (fermented swine urine) Scenedesmus sp. 6f 0.9i 0.54i Kim et al. (2007)

J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25

2 1
Agricultural (anaerobically digested dairy manure) Mix of Microspora willeana, Ulothrix zonata, Ulothrix 5.5 g m day nd nd Wilkie and Mulbry (2002)
aequalis,
Rhizoclonium hieroglyphicum, Oedogonium sp.
2 1
Agricultural (swine effluent, maximum manure loading rate) R. hieroglyphicum 10.7 g m day 0.7i 72i mg m 2 day 1 Mulbry et al. (2008)
2 1
Agricultural (dairy effluent + CO2, maximum manure loading R. hieroglyphicum 17.9 g m day 1.2i 210i mg m 2 day 1 Mulbry et al. (2008)
rate)
Agricultural (digested dairy manure, 20 dilution) Chlorella sp. 81.4g 13.6i 11i Wang et al. (2010, in
press)
h
Agricultural (dairy wastewater, 25% dilution) Mix of Chlorella sp., Micractinium sp., 59 29 17 Woertz et al. (2009)
Actinastrum sp.
Industrial (carpet mill, untreated) B. braunii 34 13.20 4.5 Chinnasamy et al. (2010)
Industrial (carpet mill, untreated) Chlorella saccharophila 23 18.10 4.2 Chinnasamy et al. (2010)
Industrial (carpet mill, untreated) Dunaliella tertiolecta 28 15.20 4.3 Chinnasamy et al. (2010)
Industrial (carpet mill, untreated) Pleurochrysis carterae 33 12.00 4.0 Chinnasamy et al. (2010)
Artificial wastewater Scenedesmus sp. 126.54 12.8 16.2 Voltolina et al. (1999)

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.

21
22 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25

concentrations of lipid tend not to be observed in open pond- and total fatty acid concentration ranging from 0.141 to 0.233 g L 1
grown microalgae (Griffiths and Harrison, 2009). Furthermore, by the end of the experiment, which equates to a maximal lipid
these very high concentrations of cell lipid tend to be coupled with productivity of 11 mg L 1 day 1. The third study which also as-
low biomass particularly when induced by environmental stress sessed growth of Chlorella on dairy manure wastewater, compared
such as N or P limitation (Dean et al., 2010; Rodolfi et al., 2009), the growth of the microalgae in suspended culture and when at-
therefore the determination of lipid productivities from high bio- tached to a polystyrene foam support growth system (Johnson
mass culture conditions such as wastewater-grown microalgae and Wen, 2010). The total fatty acid content of the microalgae
are of particular interest. was similar from both growth systems (9% DW) but biomass
A number of recent laboratory-based studies where microalgae yield was superior for the attached algae. With a total fatty acid
have been grown either in small batch cultures, small semi-contin- yield of 2.59 g m 2, this gave a total fatty acid productivity of
uous cultures or bioreactors have reported reasonable lipid accu- 230 mg m 2 day 1.
mulation in wastewater-grown microalgae, ranging from low One study has taken the analysis of wastewater-grown algae
(<10% DW) to moderate (25–30% DW) lipid content, and in some one step further and examined biomass production and lipid pro-
studies this can translate to relatively high lipid productivity when ductivity under pond-scale conditions. Fatty acid composition of
coupled to high biomass (Table 1). Microalgae grown on the the benthic algae R. hieroglyphicum grown in an algal turf scrub-
untreated carpet mill wastewater, as described above, was also ber type pond system was examined (Mulbry et al., 2008). The
analysed for lipid content. The total lipid content ranged from experiment compared raw swine effluent with raw dairy effluent
12% to 18.1% per cell DW depending on species. When taking into with or without additional CO2. Total lipid content was greater in
account the calculated biomass of the cells (ranging from 23 to the swine effluent grown algae (9.3% DW), but algal productivity
34 mg L 1 day 1) and the amount of wastewater produced each was greater on the dairy effluent (up to 21.3 g DW m 2 day 1
year, this resource was estimated to give biomass ranging from compared to 10.7 g DW m 2 day 1 on swine effluent), therefore
16.1 to 28.1 tons ha 1 year 1 and an estimated lipid yield ranging greater fatty acid productivity was provided from this effluent
from 3260 to 3830 L ha 1 year 1 (Chinnasamy et al., 2010), sug- with values calculated at up to 156 mg fatty acid m 2 day 1
gesting that energy yield from this type of waste may have future (without added CO2) and 210 mg fatty acid m 2 day 1 (with
promise. CO2), compared to just 86 mg fatty acid m 2 day 1 from swine
In a recent study of municipal wastewater, batch culture effluent (Mulbry et al., 2008). This group has now performed fur-
growth of Chlamydomonas reinhardtii was assessed and shown ther experiments to demonstrate that the lipid can be effectively
to be strong in wastewater from a variety of treatment phases extracted from the harvested and dried algal samples (Mulbry
in particular from 100% centrate wastewater, and could yield total et al., 2009).
lipid content of 16.6% DW (Kong et al., 2010). When transferred
to a biocoil, the microalgae were able to grow consistently ro-
4. Potential, limitations and future needs of algal wastewater
bustly in the wastewater for 1 month. Furthermore, lipid content
biofuel production
from the biocoil-grown microalgae reached 25.25% DW, and pro-
vided biomass productivity of 2000 mg L 1 day 1 and an esti-
The high biomass productivities and in some cases high lipid
mated lipid productivity of 505 mg L 1 day 1. In addition, this
productivities that have been demonstrated in many of the re-
lipid productivity could be coupled with efficient N and P removal
viewed studies of wastewater-grown microalgae suggests that
(Kong et al., 2010). Similar levels of total lipid content have been
there is real potential in the utilisation of these high nutrient re-
observed in B. braunii grown in secondary treated municipal
sources for cost-effective biofuel production. However, there are
wastewater (17.85% DW), and interestingly these values were
some limitations that need to be addressed.
higher than when the microalgae was grown in synthetic growth
medium (yielding 10.96% DW) suggesting that the stress condi-
tions of the wastewater may be inducing an increase in lipid syn- 4.1. The need for efficient algae harvesting techniques
thesis (Orpez et al., 2009). However, the possible positive impact
of waste addition on lipid induction may not hold out for every A significant issue to be resolved in all applications with micro-
situation. An analysis of Scenedesmus growth following the addi- algae cultivation including algal-based wastewater treatment and
tion of fermented swine urine found that although growth rate algal growth for biofuels is the need for efficient and cost-effective
and dry weight of the microalgae was increased by nearly 3-fold harvesting and processing of microalgae following cultivation. The
over a 30 day growth period, total fatty acid content was signifi- small size and relatively low density of algal cells in a typical open
cantly reduced (9 mg g 1 DW in treated cells compared to pond system coupled with the need to handle large volumes means
46 mg g 1 DW in control cells) over the same period (Kim et al., that harvesting is both difficult and costly. The lack of efficient al-
2007). gal removal systems is the major reason why algal-based wastewa-
Three recent studies have indicated the biofuel production po- ter treatment is not used extensively by the wastewater industry,
tential of algae grown on dairy manure. Woertz et al. (2009) deter- and is a significant impediment to the harvesting of biomass from
mined the lipid content from mixed algae cultures, originally high-rate algal ponds (de-Bashan and Bashan, 2010). Currently al-
isolated from local wastewater treatment ponds, and grown in gal biomass is harvested by centrifugal or gravity sedimentation or
anaerobically digested dairy manure wastewater in outdoor batch by filtration, all of which may be preceded and eased by a floccu-
cultures. After 6 days growth, peak lipid accumulation reached lation step.
14% to 29% DW depending on the wastewater concentration Flocculation is the first stage in the bulk harvesting process and
used, giving estimated lipid productivity of 2.8 g m 2 day 1 (or is used to aggregate the cells, so increasing their effective particle
17 mg L 1 day 1). Wang et al. (2010, in press) likewise assessed size and thus easing subsequent centrifugation, filtration or sedi-
batch culture-grown microalgae (Chlorella sp.) on anaerobically di- mentation steps (Molina Grima et al., 2003). Algal cells carry neg-
gested dairy manure, and performed a detailed analysis of the lipid ative charge which prevents their aggregation in cells suspension,
profile of the cells. Total fatty acid content including neutral lipids however the addition of metal salts such as FeCl3 can reduce or
(esters and triacylglycerol), phospholipids and glycolipids, ranged eliminate this charge, leading to the aggregation of cells. Alum
from 9% to 13.7% DW depending on the wastewater concentration (Al2(SO4)3) in particular has been widely used in the wastewater
used, with cell dry weight biomass ranging from 1.47 to 1.71 g L 1 industry for this purpose (Molina Grima et al., 2003). Alternative
 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25 23

flocculation methods include the use cationic polymers such as processing, conversion and oil extraction, and the need for
chitosan, or the addition of alkalis to increase the pH of the algal improvements in this part of the algal biofuel production process
media (Brennan and Owende, 2010). have been discussed extensively in other articles and will not be
Chemically-induced flocculation is however expensive. An reviewed here (Amin, 2009; Brennan and Owende, 2010).
alternative harvesting approach is the use of the so-called auto-
flocculation or bio-flocculation whereby many unicellular algal 4.2. The need for life cycle analysis of wastewater-derived algal biofuel
species spontaneously aggregate and settle to the bottom of the
pond and can then be harvested (Schenk et al., 2008). Furthermore, A critical determinant as to the true potential of algal biofuel
bio-flocculation may be induced in some species by carbon limita- production using wastewater resources is whether the process will
tion or environmental stimuli such as O2 deprivation. Bio-floccula- provide a positive energy output which will also determine the
tion of the wastewater tolerant C. minutissima was found to be very economic viability of the process. In addition there will be a need
high for 21-day cultures, although this was tested in synthetic to determine whether the process is truly sustainable in terms of
medium rather than wastewater (Bhatnagar et al., 2010). Whether the utilisation of natural resources and whether the process is truly
bio-flocculation can be efficiently induced in wastewater medium carbon neutral. Results from recent life cycle energy assessments
requires further assessment. of biofuel feedstocks including microalgae and in comparison to
Following flocculation biomass can be further harvested by cen- other bioenergy feedstocks have provided mixed conclusions (Cla-
trifugal or gravity sedimentation. The latter is the most common rens et al., 2010; Lardon et al., 2009). Lardon et al. (2009) and Cla-
technique used in wastewater treatment because of the large vol- rens et al. (2010) have both performed life cycle assessments of
umes treated and low value of the biomass generated (Molina Gri- biofuel production from microalgae in comparison to other plant-
ma et al., 2003). However, the small size of the microalgae and based biofuels and fossil fuel. Both assessments concluded that
their low specific gravity results in a settling rate that is too low based on current technology parameters, algal-based biofuels had
for routine algal harvesting. Centrifugation is therefore the pre- a positive energy balance but performed relatively poorly com-
ferred method for recovery of algal cells as it is rapid, works for a pared to other biofuels. Clarens et al. (2010) suggested that
wide range of differing microalgae, and can give high cell harvest plant-based biofuels have lower energy use, GHG emissions and
efficiency (>95%), although a major disadvantage is that it is an en- water use than algae biofuels. It was concluded that a major factor
ergy intensive process. in the poor environmental impact of algal biofuels as modelled in
A further method of harvesting is filtration. Filtration using this assessment, was the demand for CO2 and fertiliser as a nutri-
pressure of vacuum, together with the use of filter aids such as dia- ent source. Wastewater use could offset these nutrient and CO2 de-
tomaceous earth or cellulose are suitable for the recovery of larger mands. As well as being high in nutrients, many wastewaters,
algae (>70 lm) but not for smaller microalgae such as Scenedesmus particularly municipal and agricultural wastewaters, have signifi-
and Chlorella (Brennan and Owende, 2010). For small cells, mem- cant aerobic bacterial populations which will generate CO2 through
brane microfiltration and ultrafiltration are alternative methods, respiration and which can be utilised by the microalgae (Munoz
however, the need to frequently replace membranes and the cost and Guieysse, 2006). Clarens et al. (2010) further modelled algal-
of pumping makes this an expensive process. In general, centrifu- based biofuel production when coupled to three types of municipal
gation remains the most reliable and preferred method for algal and agricultural waste as sources for N and P. These models as-
harvesting, and is only slightly more expensive than alternative sumed the addition of wastewater-derived nutrient to algae in a
methods (Olaizola, 2003). freshwater-based pond. All three models significantly improved
An alternative method for harvesting microalgae is immobiliza- the life cycle burden on the algal biofuel production and when
tion of the microalgae prior to and during cultivation so that the source-separated urine was modelled as the wastewater resource,
biomass can be easily retrieved at the end of the growth period. the algal-based process was shown to be more environmentally
This harvesting method is particularly useful for water treatment beneficial than terrestrial plant biofuel crops.
applications, and various methods for this have been developed Further life cycle analysis is needed to assess the alternative
and evaluated for their efficiency of pollutant removal from waste- wastewater strategies reviewed here, which include using waste-
water (reviewed by de-Bashan and Bashan, 2010; Mallick, 2002). water resources not only as an added nutrient supply but also as
Artificial attachment or polymer encapsulation of microalgae, such the pond medium, and potentially as the driver for the energy in-
as in alginate, are the most frequently used immobilization ap- put and additional CO2 input (Fig. 1).
proaches. An effective method must maintain live cells for as long
as possible and allow high flow of the waste medium into the poly- 4.3. The need to demonstrate high biomass and lipid productivity at
mer-algae matrix to allow high nutrient accumulation by the cells. pond scale
Alginate-immobilized microalgae have been shown to be as effec-
tive as free-swimming microalgae in accumulating N and P from As described above, microalgae biomass can be processed and
municipal wastewater. For example, C. vulgaris immobilized in converted to biofuel by a variety of methods, however, lipid extrac-
alginate pellets placed in a fluidized bed column could effectively tion from microalgae and the use of microalgae as an oil source for
remove approximately 80% of the ammonia content and 70% of biodiesel production is likely to be one of the most attractive op-
the total P content in a sewage culture (Travieso et al., 1992). Sim- tions, particularly if the remaining residual algal biomass is utilised
ilarly, Scenedesmus immobilized in alginate sheets could effectively for biogas production (Brune et al., 2009). Although most microal-
remove ammonia and orthophosphate from secondary treated gae have relatively low total lipid content per cell under wastewa-
effluent (Zhang et al., 2008). Algal biomass can be recovered from ter conditions (Table 1), the potentially high biomass productivity
an immobilized matrix and this may be suitable feedstock for will translate to significant total lipid productivity. Some of the ini-
downstream thermochemical processing such as pyrolysis or for tial studies of wastewater-grown microalgae have reported very
the generation of bioethanol by fermentation. However, it is un- significant biomass yield (e.g. Kong et al., 2010) and suggest much
clear whether immobilized algae could be used for oil production promise but future work must demonstrate similar yields on a lar-
and whether lipids could be efficiently removed from an algal– ger scale and in open pond conditions. Most of the studies to date
polymer matrix. have analysed microalgae biomass and lipid production under lab-
Following harvesting, the algae have to go through a number of oratory conditions and following microalgae cultivation in batch
further processes, however the issues of downstream microalgae culture flasks or bioreactors rather than in ponds. There is also a
24 J.K. Pittman et al. / Bioresource Technology 102 (2011) 17–25

need to demonstrate that these biomass yields can be maintained Brennan, L., Owende, P., 2010. Biofuels from microalgae – a review of technologies
for production, processing, and extractions of biofuels and co-products. Renew.
over long cultivation periods. There is also the possibility that no-
Sust. Energy Rev. 14, 557–577.
vel technologies such as genetic manipulation of microalgae, which Brune, D.E., Lundquist, T.J., Benemann, J.R., 2009. Microalgal biomass for greenhouse
is being used to attempt to improve algal lipid content (e.g. Wang gas reductions: potential for replacement of fossil fuels and animal feeds. J.
et al., 2009), could likewise be used to improve algal growth and/or Environ. Eng. 135, 1136–1144.
Canovas, S., Picot, B., Casellas, C., Zulkifi, H., Dubois, A., Bontoux, J., 1996. Seasonal
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An alternative method to improve the lipid productivity of rap- Water Sci. Technol. 33, 199–206.
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