Bioresource Technology 107 (2012) 19

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

Review

Integrated green algal technology for bioremediation and biofuel

Ganapathy Sivakumar a,, Jianfeng Xu a, Robert W. Thompson b, Ying Yang c, Paula Randol-Smith d,
Pamela J. Weathers c
a

Arkansas Biosciences Institute, College of Agriculture and Technology, Arkansas State University, Jonesboro, AR 72401, USA
Department of Chemical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA
c
Department of Biology/Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA
d
Green Wisdom Inc., Cotton Plant, AR, USA
b

a r t i c l e

i n f o

Article history:
Received 20 August 2011
Received in revised form 29 November 2011
Accepted 16 December 2011
Available online 24 December 2011
Keywords:
Biodiesel
Jet fuel
Hydrocarbon
Lipid
Sustainability

a b s t r a c t
Sustainable non-food energy biomass and cost-effective ways to produce renewable energy technologies
from this biomass are continuously emerging. Algae are capable of producing lipids and hydrocarbons
quickly and their photosynthetic abilities make them a promising candidate for an alternative energy
source. In addition, their favorable carbon life cycle and a renewed focus on rural economic development
are attractive factors. In this review the focus is mainly on the integrated approach of algae culture for
bioremediation and oil-based biofuel production with mention of possible other value-added benets
of using algae for those purposes.
Published by Elsevier Ltd.

1. Introduction
Petroleum reserves are globally nite and indeed peak oil production may already have occurred (Kerr, 2011), so efforts to develop alternative sustainable energy sources are under aggressive
global development. Algae are one of the proposed platforms for
renewable energy production that is not likely to impact agriculture (Pittman et al., 2011). Photosynthetic algae use energy from
the sun to grow rapidly accumulating biomass that contains proteins, lipids, hydrocarbons, carbohydrates, small molecules and
pigments. Microalgae also consume large amounts of nitrogen,
phosphate, and CO2 that are converted into biomass thereby making these species attractive for CO2 mitigation and pollution reduction of waterways, even for reduction of toxic chemicals (Kumar
et al., 2011; Park et al., 2011). Some species of microalgae can produce >50% of their dry mass as triacylglycerols or long chain hydrocarbons that can be converted to biodiesel and jet fuel or cracked
like petroleum for liquid fuel (Sivakumar et al., 2010; Niehaus
et al., 2011). Microalgal biomass also can be processed into other
biofuels through biophotolysis to biohydrogen, anaerobic digestion
to methane, and via thermochemical processes into bio-oil, syngas,
charcoal and electricity. Macroalgal species accumulate signicant
amounts of sugars that have a low lignin content which can be fermented into bioethanol or butanol (Jones and Mayeld, in press).
Corresponding author. Tel.: +1 870 680 4809; fax: +1 870 972 2026.
E-mail address: sivakumar@libero.it (G. Sivakumar).
0960-8524/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.biortech.2011.12.091

Moreover, microalgae-derived bio-based products can also be used

in the production of cosmetics, food or food additives, and fertilizers. Some marine algal species (e.g., Haematococcus pluvialis, Emiliania huxleyi, Dunaliella tertiolecta) have been optimized for
overproduction of products including astaxanthin, b-carotene,
omega-3-fatty acid, vitamin E and other pigments (Lohr et al., in
press; Pangestuti and Kim, 2011; Sayanova et al., 2011). Thus far,
more than 20 recombinant proteins have been successfully expressed in microalgae as summarized in a recent review on this topic (Potvin and Zhang, 2010). Integrated algal systems can be used
for wastewater treatment and bioremediation to capture carbon,
nitrogen and phosphorus from specialty industrial, municipal and
agriculture wastes (Fig. 1). Algae are therefore an attractive biofactory for establishing a sustainable community such as the one
envisioned for Cotton Plant, AR where Green Wisdom Inc. plans
to implement an integrated algal production system to recycle
agricultural wastes for biofuel (see Section 6).

2. Algae for bioremediation

2.1. Phosphorus and nitrogen removal
Although nitrogen and phosphorous are elements key to algal
growth, they are also serious pollutants in many waterways. Algae
can thrive in nitrogen- and phosphorus-rich conditions common to
many wastewaters (Pittman et al., 2011), and this feature may be

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

CO2

Solar energy

Filter
N, P
Power plant
Biomass harvest

Biobased
products

Oil extraction
Agricultural or
municipal waste water
Anaerobic digester
effluent

Algal culture
TAGs

Hydrocarbons

Transesterification

Hydrotreating

Hydrocracking

Biodiesel

n-Alkanes

Jet fuel, gasoline

Waste water

Bioremediation

Biofuel Production

Fig. 1. Schematics of an integrated algal culture system for bioremediation and biofuel production.

harnessed to not only remove, but also capture these important

nutrients in order to return them to the terrestrial environment
as agricultural fertilizer. This could provide a high value by-product for algae that are primarily being grown for biofuel. Several
bioremediation options related to capture and retrieval of these
two key nutrients are presented here.
2.1.1. Phosphorus
Phosphorus is critical to agriculture, yet the worlds supply of
readily available rock phosphate reserves is dwindling and global
supplies are estimated to be depleted in 80100 yr (Abelson,
1999) and by 2033, the demand for phosphate is projected to outpace supply. Phosphorus is often limited in soils because it may
form insoluble complexes with cations, and tropical soils are especially susceptible to phosphorus deciency (Vance et al., 2003).
Water-soluble phosphate, when applied as fertilizer, is often lost
from soils and <20% is absorbed by plants (Vance et al., 2003).
Phosphate then enters ground water, streams, and rivers and
moves out to sea where 100 Gt of phosphorus reside and are
essentially lost (Baturin, 2003). Most of this phosphorus enters
the oceans from river runoff with 20 Mt yr 1 as suspended phosphorus, and 1.5 Mt yr 1 as dissolved forms of phosphorus; however, marine phytoplankton consume from 1.5 to 2.5 Gt yr 1 of
dissolved phosphorus (Baturin, 2003). Acid rain has only exacerbated the problem because phosphate is highly soluble at pH < 7.
Polyphosphates have been found in nearly all forms of life
including micro- and macro-algae which accumulate this polymeric form of phosphorus as polyphosphate bodies (PPB) stored inside an organelle, the acidocalcisome (Seufferheld and Curzi, 2010).
In some microbes and under certain conditions, PPBs nearly ll the
cell (Khoshmanesh et al., 2002). In microalgae, accumulation of
polyphosphate occurs when phosphorous becomes available to
phosphorous-starved cells (over-compensation or overshoot) or
even when cells are not starved for this element (luxury uptake)
(Powell et al., 2009). More detailed knowledge of the regulation
of phosphate uptake by algae is required for their effective use in
the recovery of phosphorus from stream and river runoff or from
growth on waste streams, e.g., feedlots. For recent reviews on polyphosphate in photosynthetic organisms see Yang and Finnegan
(2010) and Seufferheld and Curzi (2010).
2.1.2. Nitrogen
Algae remove nitrogen more effectively than phosphorus from
their environment and thus decrease the nutrient levels and improve water quality (Aslan and Kapdan, 2006). Although nitrogen

is essential for a healthy ecosystem, in excess this nutrient is a pollutant, which is responsible for such events as toxic algal blooms,
loss of oxygen, and loss of biodiversity (Carpenter et al., 1998).
Controlling the nitrogen content of bodies of water and waterways
is difcult because there are a variety of inputs. Variations in soil
composition, species of grazing animals, nitrogen levels in fertilizers used for agricultural and residential activities play major roles
in the degree of water contamination (Bryant et al., 2011). Plants,
microbes and algae absorb nitrogen from soil or water and store
it as biomass. Over time the biomass decomposes releasing the
nitrogen back into the soil (e.g., as ammonia, urea) or atmosphere
(e.g., as N2O), where it may be recycled or lost (Woodward et al.,
2009). Photobioreactors harboring immobilized algae are being
developed to effectively remove nitrogen from different sources
(Kapdan and Aslan, 2008).
When grown in the presence of nitrite, some microalgae and
cyanobacteria can produce N2O (Weathers, 1984; Weathers and
Niedzielski, 1986). Although this gas was not produced in signicant amounts in the presence of nitrate, it is not known if other
nitrogenous compounds found in wastewater such as urea and
ammonia are converted to this potent greenhouse gas. Measuring
the production of N2O whenever an algae biofuel-CO2 remediation
system is being considered appears to be prudent.
The US is the worlds second largest user of nitrogen. Recently,
AquaFUELs (<http://www.aquafuels.eu>) estimated that the European Union (EU) alone required nitrogen and phosphorous of about
20 and 4 million tons, respectively, for substituting fossil fuels with
algal fuels. In other words, the need is double the current EU fertilizer production capacity (van Egmond et al., 2002). On the other
hand, microalgae have unique natural mechanisms for removing
excess CO2, nitrogen, and phosphorus from water sources. Although
current algal technology for fuel has a negative energy balance,
development of an integrated process for bioremediation and economic feasibility models for commercializing biodegradable algalfuels are underway. If integrated algal technology proves successful, it would not only make algal fuel competitive, but also provide
a diverse range of benecial impacts on the environment, including
carbon sequestration. Moreover, opportunities are at hand to
implement this technology in unused desert land and coastal plains
for sustainable large-scale algae production. This may also protect
agricultural topography and minimize the cost of fertilizers.
2.1.3. Linkage of algal bioreactor systems to waste streams
The nutrients required to support algal growth can be met by a
variety of sources, but pristine raw materials will likely be too

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

costly to make algal-based fuels economically competitive with

other fuels. Supplying nutrients (principally carbon, nitrogen, and
phosphorus) by using waste products would make the production
system more viable. In this way, algal cultures solve environmental
problems while simultaneously producing fuels or other useful
chemicals. For example, the National Renewable Energy Laboratory
(NREL, Golden, CO) has used efuent from a power plant to capture
CO2 emissions (Sheehan et al., 1998). While several reports indicate that costs of algal-based biofuels can be reduced by using
waste streams (Grant, 2009; Pittman et al., 2011), several authors
have suggested that fuel production using algal cultures will not
be competitive without the use of waste streams (Feng et al.,
2011; Park et al., 2011; van Beilen, 2010), and van Beilen (2010)
claimed that bioremediation will be of primary importance, while
biofuel was only a by-product of that operation. Recent economic
analyses of the production costs of algal oil have estimated that
it is competitive with petroleum at $60/barrel (Chisti, 2008) or
$64/barrel (Feng et al., 2011), but only if a credit is assumed for
the wastewater treatment component of the operations.

2.1.4. Scope of nutrient feed potential

Cost-effective production of biofuels using algae requires a signicant input of nutrients which could come from waste such as
municipal sewage and feedlot runoff (Pittman et al., 2011). These
sources are often isolated (feedlots), or in urban areas (sewage)
and it is not clear if these sources will be adequate to provide
the anticipated levels of algal oils needed despite year-round availability of large quantities. Other waste streams that have been
examined include industrial carpet mill efuent (Chinnasamy
et al., 2010), rice hulls, soy eld debris, catsh processing waste
(Yang et al., 2011), dairy and swine manure and urine (Mulbry
et al., 2008), and others (Table 1). Some of these wastes might
not be fully available for algae production as they are also used
as fertilizers (Swana et al., 2011). Although some of these wastes
may serve as individually adequate nutrient sources, it is more
likely that mixed wastes will produce a better nutrient stream as
was demonstrated by Yang et al. (2011) who showed that when
three anaerobic waste efuents were mixed and fed to Ettlia oleoabundans, there was a synergistic response; more biomass and oil
were produced than from any single waste stream or the sum of
separately fed waste streams. Some of these complex materials
will rst require anaerobic digestion to produce a simple nutrient
stream. For example, apple pomace, which is rich in residual sugars
will certainly induce heterotrophic growth and algal species selected for bio-oil production using such feedstock must demonstrate robust growth as well as oil production under these
conditions. Although not necessarily true for all species, heterotrophic conditions can yield increases in biomass and lipid contents as

shown by Wan et al. (2011) for Nannochloropsis oculata, Dunaliella

salina, and Chlorella sorokiniana. Heterotrophic growth can be benecial for remediation of liquid waste streams; however, the presence of reduced carbon is problematic in open cultivation systems
that are not axenic since fast-growing microbial contaminants that
consume preferentially consume reduced carbon will readily outpace the algal population.
Among the reasons that it is difcult to make reasonable decisions regarding the design and development of algal biofuel production systems is the inherent variation of the content of various
waste stream sources. Obviously, the carbon, nitrogen, and phosphorus contents of wastewaters will vary depending on their
source, geographic location, and perhaps season, potentially necessitating design or operational modications in production systems.
One way of avoiding the variability of using real wastewater
sources is to create articial simulated wastewater in the laboratory (Feng et al., 2011; Pittman et al., 2011) so that the effects of
each component as well as those of the total nutrient supply on algae cultures can be studied. However, these articial wastewaters
may not be completely reective of real wastewater, since minor
chemical constituents or agents such as viruses present in real
wastewaters may be absent. Therefore, it is important that real
waste streams are used in algal studies aimed at developing a commercially successful operation.
Municipal sewage, industrial, and agricultural wastewaters contain carbon, nitrogen, and phosphorus to varying degrees. Agricultural wastewater often has higher levels of nitrogen and
phosphorus than municipal sewage, and the composition of industrial wastewaters depends on the type of industry that creates
them and might even include wastewater loaded with heavy metals that would be of little value in promoting algal growth (Pittman
et al., 2011).

2.1.5. Carbon dioxide

Although some algal species, e.g., diatoms, are known to x CO2
via the C4 pathway (Reinfelder et al., 2000), most algae demonstrate C3 carbon xation via the BensonCalvin cycle. CO2 xation
is highly regulated by light intensity, temperature, and CO2 concentration. Generally more CO2 can be xed at moderate temperatures and with increasing light intensity. CO2 is nearly 30 times
more soluble in water than O2 at 20 C and 1 atm (Atkinson and
Mavituna, 1991), but the solubility of gases in liquids declines with
increasing temperature and solute concentration. When the rate of
CO2 xation is high, hydroxide ions accumulate in the medium and
the pH rises to as high as 11. Inadequate xation of CO2 by a culture can yield medium acidication from excessive carbonic acid
formation, so a rapidly growing healthy algal culture will have a
balanced pH with photosynthetic hydroxyl ions being offset by

Table 1
Examples of waste resources for consideration as nutrient feeds stocks for algae.
Waste material

Seasonality

Estimated global
amount

% of nitrogen (dry
weight basis)

% of phosphorus (dry
weight basis)

Competing uses

Apple pomace

Late summer,
fall
Summer
through fall
Spring through
fall

16  106 ta

0.57b

0.09b

Composting, ethanol, animal feed

Ethanol, animal feed, composting

Potato process
waste
Biodegradable yard
waste
a
b
c
d
e
f

6 c

3.3  10 t

0.34

0.07

31  106 te (US
only)

e.g., Lawn clippings and

leaves
3.0f

0.30f

Kennedy et al. (1999).

Linskens and Jackson (1999).
<http://www.scri.ac.uk/scri/les/IIPP%20presentations/PamelaAnderson.pdf>.
<http://www.umaine.edu/umext/potatoprogram/Fact%20Sheets/RecyclingPotato318.pdf>.
<http://www.epa.gov/ttnatw01/burn/yardburn.pdf>.
<http://www.fao.org/docrep/007/y5053e/y5053e0e.htm>.

Composting, co-generation via combustion or

anaerobic digestion

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

carbonic acid production from the entering dissolved CO2 (Xu et al.,
2009).
CO2 mitigation to combat global warming is considered one of
the prime benets of algal biofuel production. One of the best
sources of highly enriched CO2 is ue gas, which contains
1020% CO2 from burning fossil fuels (Ge et al., 2011). Algal species
differ in their apparent ability to use CO2 effectively. High level of
CO2 inhibit some species like Chlorella (Chiu et al., 2008), while
others like Botyrococcus (Ge et al., 2011) can thrive on CO2 levels
up to 20%. The provision of adequate CO2 supplies and maximizing
cost efcient mass transfer of CO2 to cells in the aqueous environment of large-scale liquid culture systems is not a trivial task, and
technology is still needed to improve mass transfer and minimize
costs.
3. Algal lipid and hydrocarbon production chemistry
3.1. Triacylglycerol and hydrocarbon metabolism
Algae can accumulate 2075% of lipids as part of their dry biomass. Most species produce triacylglycerides (TAGs) (Scott et al.,
2010) and alkanes (Radakovits et al., 2010) originating from the
fatty acid biosynthetic pathway, but a few species accumulate long
chain hydrocarbons (LCHs) originating from the terpenoid pathway (Niehaus et al., 2011). In addition to oils or hydrocarbons, algal
biomass can yield other useful products or it can be used for the
production of animal feed, bioethanol or methane. Hydrolysis of
TAGs yields free fatty acids and glycerol that can be used in the
cosmetic and pharmaceutical industries or, in the case of glycerol,
as precursor for the synthesis of various chemicals such as propylene glycol (Dasari et al., 2005) or as a carbon source for industrial
microorganisms (Vlysidis et al., 2011).
3.2. Genetic engineering options for oil improvement
Transgenics and metabolic engineering have the potential to improve not only algal growth, but also lipid productivity and the type
of lipids produced. The ultimate aims are high quality lipids with
fatty acid mixture of 16:1, 18:1, and 14:0 in the ratio of 5:4:1
(Schenk et al., 2008; Voelker et al., 1992). This ideal mix has been
identied as providing a good Cold Filter Plug Point (CFPP) rating
and cetane number along with low oxidation potential.
One way to increase cellular lipid yield is to block metabolic
pathways that lead to the accumulation of energy storage
compounds such as starch or to decrease lipid catabolism such as
b-oxidation that consumes TAGs (Radakovits et al., 2010). For
example, two different starch-decient strains of Chlamydomonas
reinhardtii with disruptions in the ADP glucose pyrophosphorylase
or isoamylase genes, respectively, have been shown to accumulate
increased levels of TAGs during nitrogen deprivation (Wang et al.,
2009). A starch-free mutant of Chlorella pyrenoidosa has further
been shown to have elevated polyunsaturated fatty acid content
(Ramazanov and Ramazanov, 2006). In another example, the fatty
acid chain length was altered in Phaeodactylum tricornutum by
overexpression of 12:0-biased thioesterase (from Umbellularia californica) and 14:0-biased thioesterase (from Cinnamomum camphorum) (Radakovits et al., 2011). Another successful approach for
engineering algae to increase biomass yields is to down-regulate
expression of the light-harvesting antenna complexes (LHC). When
LHC expression was down-regulated in transgenic C. reinhardtii,
greater resistance to photooxidative damage and up to 50% increase in photosynthetic efciency were observed (Beckmann
et al., 2009; Mussgnug et al., 2007). This modication enabled algae to tolerate higher light intensities that may otherwise be inhibitory. Considering that the typical light tolerance of algae in

photobioreactors is 200400 lmol m 2 s 1, about 1020% of photosynthetically active radiation (Gordon and Polle, 2007), improvement in LHC-related photosynthetic efciency should improve
algal productivity.
Although the application of genetic engineering to improve biofuel production in algae is in its infancy, signicant advances have
been made in the development of genetic manipulation tools in the
past years. Many companies such as Synthetic Genomics (La Jolla,
CA; <http://www.syntheticgenomics.com>), Solazyme (South San
Francisco, CA; <http://www.solazyme.com>), Sapphire Energy (San
Diego, CA; <http://www.sapphireenergy.com>), Phycal (Highland
Heights, OH; <http://www.phycal.com/site>), Seambiotic (Israel;
<http://www.seambiotic.com>) and TransAlgae (Israel; <http://
www.transalgae.com>) are working on genetic engineering of algae.
For example, Synthetic Genomics is engineering microalgae to continuously excrete oils from the cells, which will allow the non-invasive extraction of algal oils. It seems as if genetic engineering is the
only practical solution to create super algal strains with all the
attributes suitable for biofuel production; however, if these genetically engineered algae are not constrained, they could pose a threat
to the environment (Beer et al., 2009; Yoon et al., 2011).
It is possible, and perhaps inevitable, that GM algae grown in
open ponds or enclosed bioreactors may escape human control
and replicate in the environment. To overcome this problem, engineering algae that have suicide genes or require a controlled
environment for growth has been proposed by Craig Venter from
Synthetic Genomics (Exploring Algae as Fuel, <http://www.heraldtribune.com>). The Israeli company TransAlgae is creating algae
with traits that paralyze them in the wild; Sapphire also claims
that its algae will be unequipped to live outside a controlled environment; and Phycal is developing a bio-security system in algae
(Are Genetically Modied Algae a Threat?, <http://www.renewableenergyworld.com>). To avoid regulatory issues faced by genetically engineered algae, algal mutants with desirable traits can be
selected. For example, the cw15 sta6 mutant of C. reinhardtii produces twice the level of TAGs as the cw15 strain when starved of
nitrogen possibly due to rerouting of carbon from starch to TAGs
(Wang et al., 2009).
3.3. Light and temperature effects
Different algal species respond differently to variations in light
and temperature encountered in outdoor closed photobioreactors.
In response to light changes in the environment, algae can initiate
photoacclimation (a physiological process) or photoadaptation (an
evolutionary process) (Falkowski and La Roche, 1991). Photoacclimation is carried out by adjusting the synthesis and degradation
level of the light-harvesting complex in the photosynthetic
machinery (Falkowski and La Roche, 1991). Under sub-saturating
light intensity, the assembly of light harvesting complexes in algae
is stimulated with a detectable increase in pigments such as chlorophyll a and photosynthetic reaction center proteins such as D1
and D2 (Hoffmann and Senger, 1988). Oversaturating irradiance
triggers the degradation of photosynthetic pigments and proteins
while increasing accumulation of secondary carotenoids to protect
the reaction center from excess light exposure (Richmond, 2004).
Carbon ux in algae is then redirected from the primary products
of photosynthesis, simple sugars, to polysaccharide and/or lipid
synthesis (Friedman et al., 1991). Meanwhile, the cell quota of light
harvesting complexes can also be decreased by stimulated cell
division (Richmond, 2004). Excessive light exposure can severely
damage the algal photosynthesis machinery and should be avoided
in algal cultures.
Variations in spectral quality can also alter the physiological
status of algae. The wavelengths of maximum absorption are different in the two photosystems (PS) 680 nm for PS II and

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

700 nm for PS I. Therefore, the excitation of PS II requires mostly

red light while PS I requires red and far red light. Different light
quality ratios such as red:far-red and blue:red may result in modied pigment compositions and photosystem II/I ratios (Rdiger
and Lo9 pez-Figueroa, 1992). Additionally, effects of light on algae
can be controlled by photoreceptors that are independent of the
photosynthetic apparatus (Hegemann, 2008). For example, though
the existence of phytochrome in microalgae has not been fully conrmed, some short-time red/far-red reversible effects have been
observed in microalgae like Dunaliella and Chlorella (Ruyters,
1988; Kreslavsky et al., 1997). For bioreactor and eld mass cultures, light is the main limiting factor due to its poor penetration
into dense cultures. Though green light (500600 nm) represents
the least favorable wavelength compared with blue (400
500 nm) and red (600700 nm) lights, its penetration depth is 20
times greater than the other two wavelength ranges (Richmond
and Cheng-Wu, 2001). How to better use green light in dense algal
cultures remains an interesting and challenging question.
Changes in light intensity and quality can alter biofuel quality.
Incident light intensity can be negatively correlated with the proportion of polyunsaturated fatty acids in the total lipids of algae
(Sukenik et al., 1989), but high light exposure has also been observed to increase the volumetric content of arachidonic acid
(C20:4) in Parietochloris incisa (Solovchenko et al., 2008). Far-red
light inhibited cell growth and photosynthetic activity, but stimulated carotenoid accumulation in Dunaliella (Snchez-Saavedra
et al., 1996). Katsuda et al. (2006) showed that red LED supplied
light greatly enhanced algal growth, while blue light enhanced
astaxanthin production. If shown to be cost effective, the use of
LED-optical ber illumination systems may prove useful for commercial scale photobioreactor culture of algae (Chen et al., 2011).
As with light, the effect of temperature on growth and productivity is species specic. For example, Chlorella strain MFD-1
showed signicantly greater growth rates and cell densities than
Nannochloropsis strain MFD-2 at all temperatures tested from
1535 C (James et al., 1989). Studies of four tropical microalgae
revealed that Isochrysis sp. and a commercial species Tahitian
Isochrysis sp. (T.ISO) showed very poor growth at 35 C, while Nitzschia closterium barely grew at temperatures >30 C or <20 C. N.
paleacea grew slowly, but achieved high lipid content at 10 C (Renaud et al., 1995). Increased production of photosynthetic enzymes
occurs at sub-optimal temperatures to maintain the effectiveness
of light harvest and electron transfer, but when the temperature
is well above the optimum, formation of carotenoids may take
place in response to formation of active oxygen radicals and problems of heat dissipation (Richmond, 2004). Changes in temperature
also bring certain side effects such as alteration of nutrient uptake
rate and algal size. Algae tend to maintain minimal cell size at optimal temperatures (Richmond, 2004).
Normally, the degree of unsaturation of algal membrane lipids
increases if cultures are maintained at temperatures below their
optimum. Unsaturated lipids ensure the stability and uidity of
cellular membranes to effectively protect the reaction center of
the photosynthetic machinery (Richmond, 2004). For example,
the total x3 fatty acid content in Chlorella strain MFD-1 increased
when temperature decreased from 35 to 15 C (James et al., 1989).
None of the four tropical algae investigated by Renaud et al. (1995)
showed high levels of polyunsaturated fatty acids (PUFAs) at high
temperatures.

4. Towards large-scale algal culture

Chlorella, Dunaliella, and Spirulina, are commercially produced at
levels of about 1000 tons per year mainly in open raceway ponds
(Grima et al., 2010). However, these systems are fraught with prob-

lems including lack of control, contamination, loss of water by

evaporation, and irregular productivities. Protozoa and zooplankton graze on algae; and fungal parasites and viral infections reduce
overall productivity. To minimize wild algal contaminants, closed
photobioreactor systems were established to provide a more predictable and consistent yields of algal biomass, oil and small molecules, but during winter or within temperate regions, operating
cost-effective photobioreactors is challenging because of the electricity (heating power/temperature control) costs.
4.1. Photobioreactor designs
Different geometries of indoor and outdoor photobioreactors
have been proposed for algal biomass production (Kumar et al.,
2011). The main designs include bubble columns, at plates, annular column, and tubular reactors. These designs are described in detail in several recent reviews (Posten, 2009; Xu et al., 2009). Some
of the more recent advancements include use of a water-embedded reactor, the Lumian AGS4000 developed by Solix Biofuels
(Fort Collins, CO, USA), that lowers capital costs by using plastic
bags that hang vertically in a well of water. The surrounding liquid
serves to buffer temperature, provide some support, and diffuse
light all around the bag (<http://www.solixbiofuels.com>). Proviron also uses a similar design with hanging slim panels of plastic
culture bags inside a large transparent bag lled with water. The
entire portable system, the ProviAPT photobioreactor, can be rolled
out and once lled with the surrounding water, support the suspended culture bags (<http://www.proviron.com/product-groups/
microalgae/proviapt-photobioreactor>). Another development by
Subitec GmbH (Stuttgart, Germany) uses a at-panel airlift photobioreactor that includes a repeat static mixer that, along with upward gas sparging, insures an averaged optimum exposure to light
(<http://www.en.subitec.com>). The at panels are constructed of
two deep drawn molded transparent plastic sheets that are fused
to one another and include the static mixers. All of these systems
are relatively low cost, sterilizable and readily scalable. Actual productivity data for long term production of algae from these systems are still pending.
4.2. Harvest and oil extraction
Generally, centrifugation and occulation have been proposed
to concentrate algae from their growth medium, but these methods are not very cost effective when producing a low value product
like biofuels. Although achieving high biomass yields of about
10 g L 1 can help offset harvest costs, most phototrophic cultures
do not achieve that density (Morweiser et al., 2010). To further reduce costs, producing secreted high-value molecules can provide
another income stream (Ho et al., 2011). Relatively easy harvesting
should also be possible by growing algae attached to a support,
such as polystyrene, and scraping cells off the support. Since not
all cells are removed, those remaining act as inoculum for the next
round of biomass and oil production (Johnson and Wen, 2010). This
approach has achieved dry biomass and oil yields of 25.65 g m 2
and 2.31 g m 2, respectively.
Once the harvesting phase has been successfully completed,
other challenges in biodiesel production arise during oil extraction
and subsequent transesterication to biodiesel. Extraction of lipids
from harvested cells is a complex and relatively costly procedure,
and while there are current methods available, improved efciency
is needed. The use of solvents, dewatering of cells, and the technological shortcomings therein provide additional challenges to cost
effective production of algal biodiesel.
A breakthrough may be on the horizon as described by Sheehan
(2011) who modeled several algal oil production methods including one developed by VG Energy (<http://www.vgenergy.net/

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

links_pr032511.php>). Using their metabolic disruption technology originating from cancer work by their parent company Viral
Genetics, a lipid trigger was used to induce algae to not only increase stored lipids threefold, but to also release their lipids into
the culture medium. This could result in lipid recovery by otation
with most of the algal cells being recyclable, eliminating the need
to harvest cells and extract lipids thereby signicantly reducing
process costs.
Taking a truly green approach, Scragg et al. (2003) concentrated
oil-forming Chlorella cells and mixed them with transesteried rapeseed oil and a surfactant for use as fuel in a diesel engine.
Although there was a slightly lower caloric value to the algal
mix, exhaust emissions compared favorably with pure rapeseed
biodiesel. However, this novel technological approach has not progressed further.

5. Energetics of using algae do algal biofuels make sense?

Two metrics have been used as measures of the efciency (or
economic value) of alternative energy processes, net energy balance (NEB) (Frondel and Peters, 2007; Hill, 2007), also expressed
as energy return on energy invested (EROI) (van Beilen, 2010),
and the net energy ratio (NER), also expressed as the fossil energy
ratio (FER) (Xu et al., 2011). The NEB (or EROI) is simply the difference between the energy in the biofuel product and the energy
needed to run a process; the more positive the difference, the better result. Results are typically given in units of MJ/L of biofuel
product. The NER (or FER) is the ratio of the same numbers, i.e.,
the energy contained in the fuel divided by the energy required
to run the biofuels process. The results are dimensionless; values
higher than 1.0 are desirable, the higher the better.
The energy content of a biofuel must be considered for the use
of that biofuel. When hydrocarbons are burned completely, the
principle products are CO2 and H2O. However, the H2O leaves the
combustion zone as a vapor, which in some circumstances may
condense to liberate its latent heat of vaporization. If the biofuel
is used to provide heat (say in a home) the water vapor may well
condense, giving up its latent heat of vaporization in the form of
heat. In those circumstances using the high heat value, HHV, in
NEB or NER calculations is appropriate. However, when used as a
transportation fuel the water vapor does not condense in the engine. In those cases, the low heat value, LHV, must be used in
NEB or NER calculations. This issue has been discussed previously
(Akinci et al., 2008) in the context of prior work that used the HHV
in NEB analyses (Hammerschlag, 2006).
Differences in NEB or NER calculations between research groups
can occur because of differences in what was included in the lifecycle analyses (chemical processing, agricultural energy, transportation, energy credits for byproducts, etc.). These details are
especially well articulated in Hill (2007). Results may vary depending on the agricultural oil used to produce the biodiesel (Akinci
et al., 2008) and depending on the particular chemical process selected for conversion of the bio-oil to biodiesel, such as transesterication or thermal depolymerization. Variations in the predicted
benets of biodiesels may also arise when different species are
used or even when the same species is grown under non-identical
conditions in different studies. For example, biomass productivities
have been reported to be in the range of 573 g m 2 day 1 for
eleven algal samples (van Beilen, 2010). Productivities also may
vary with nutrient sources, which may not be properly accounted
for in NEB or NER calculations. For example, dry weight (DW) biomass productivities range between 6 and 17,900 mg L 1 day 1, and
lipid contents range between 0.731.4% of DW when various
strains of algae were grown on municipal, agricultural, or industrial wastewaters (Pittman et al., 2011). The oil content of eight

microalgae was reported to range between 1577% of DW (Wen

and Johnson, 2009). Lastly, it has been noted that productivities
in open outdoor ponds can be signicantly lower than in laboratory environments, perhaps due to invasion by air-borne species
or other toxins (Grant, 2009; Sheehan et al., 1998). All of these factors may result in variations in NEB or NER calculations, and
assumptions regarding these need to be articulated by research
groups. While NEB analyses are easy to nd for soybean biodiesel,
those for algal biodiesel are rather few in number (Xu et al., 2011).
Lardon et al. (2009) conducted a life-cycle assessment for dry
and wet extractions, with normal nitrogen (46 g kg 1) and lownitrogen (10.9 g kg 1) wastewater feeds. Both fertilizer and energy
requirements were lower for the low-nitrogen culture condition.
Heating requirements were also lower using the wet oil extraction
process. Consequently, the wet extraction and low-nitrogen methods were the best of the four scenarios tested. An NER value was
not computed, but for 1 kg of algae biodiesel produced this condition required 19.8 MJ of energy and produced 86 MJ in the biodiesel; yielding an NER = 4.34. The authors suggested that decreasing
fertilizer and energy consumption would make the process more
favorable.
Xu et al. (2011) developed energy balance analyses for dry and
wet routes for oil extraction, and for a standard nitrogen
(0.150 kg m 3) and a low nitrogen (0.049 kg m 3) wastewater feed.
The authors reported that if waste heat from a nearby power plant
was used to supplement the energy requirements of the algae processes, NER values of 2.38 and 1.82 could be obtained for the dry
and wet extraction processes, respectively. However, their denition of NER involved the HHV for the biodiesel, and is unlikely to
apply to using the biodiesel in the transportation sector. Consequently the actual NER values for the biodiesel used as a transportation fuel will be slightly less than the reported values.
A study of algal growth on simulated wastewater computed an
NER value of 1.25 for C. vulgaris (Feng et al., 2011), a result that
demonstrated that the net energy contained in the algal biodiesel
was modestly higher than the energy required to produce the oil.
The value is likely quite close to the values Xu et al. (2011) would
have computed using the LHV.
In a thorough life-cycle analysis of a raceway algae production
facility, Campbell et al. (2011) analyzed 0.71 m deep ponds with
a single paddle wheel circulator. The culture was assumed to grow
with a local CO2 source in sea water. The algae would be harvested
with a chemical occulant, the lipids converted by transesterication, and anaerobic digestion of the algal biomass would be used to
produce a combustible gas to generate electricity. Cost of peripheral road drainage, buildings, and other miscellaneous energy expenses were also included. The authors reported the results of
their energy balance in terms of Articulated Truck MJ tonne-kilometer 1 for ideal and realistic conditions. While the ideal
results yielded a very favorable positive energy balance, the realistic case was somewhat less, but still favorable.

6. An example of how a sustainable community could be

developed that incorporates algal biofuels
The process begins with an evaluation of resources to identify
those local organic waste streams that can be used to produce energy and valuable commodity products, by-products, and employment services (Christenson and Sims, 2011). The resulting products
and services will then meet the communitys unique energy and
employment needs while creating the least environmental carbon
footprint attainable as the community grows. Thus a customized
locally planned design will have to be created that is compatible
with algal growth for energy and fuel. As example, in 2006, Green

G. Sivakumar et al. / Bioresource Technology 107 (2012) 19

Fig. 2. Schematic illustrating G-WISP, a concept design that incorporates algal cultures as a part of a sustainable community planned for rural Arkansas. Copyright
(TX0007439665, 2008) permission to print this gure has been granted by the owner and co-author, Paula Randol-Smith, Green Wisdom Inc., on 11/29/2011.

Wisdom Inc. developed the Green Wisdom Inc. Sustainability Plan

(G-WISP; Fig. 2).
Communities of the future can create processing facilities that
support algae production as their common core goal by developing
local sustainable systems, like G-WISP. This would mean using
existing crops or rotational crops which are desirable for their
waste-to-energy value. Using local, multiple mix agricultural
waste and alternative technologies in near closed-loop systems,
these communities can create cost effectiveness and produce jobs.
Algal production, combining and integrating alternative energy
technologies, will foster synergistic development supporting a
self-sustaining community system. This system, represented by
the G-WISP schematic (Fig. 2), uses anaerobically digested agricultural waste materials including catsh processing waste to feed algal cultures. The illustration shows multiple environmental
abatements of CO2, e.g., from the anaerobic digester, and reclamation and use of other waste streams, such as water and heat, e.g.,
from the digester and co-generation, to support optimum sustainable algal growth. Further, the illustration demonstrates how nearclosed-loop sustainable systems can create products, algal oil and
methane energy, and by-products, e.g., fertilizer, and even multiple
resulting business spin-offs, companies to help market or distribute the products and byproducts, which, in turn, create jobs for
communities of the future. This sustainable community plan
would insure rural revitalization and, ultimately, global economic
development, while curbing the US dependence on foreign and
domestic fossil fuels.

7. Conclusions
Improving algal biomass and lipid or hydrocarbon productivity
and generating cost-effective upstream/downstream processes are
currently challenging because of the large genetic diversity of algae. If signicantly improved, algae could provide one of the new
generations of alternative clean energy. A successfully integrated

algal system could reduce green fuel costs and may provide valuable by-products thereby making algal fuels cost competitive. Likewise, algal production systems may minimize the negative effects
of CO2 and water pollution. As technology improves, so also do the
opportunities for achieving more sustainable living.
Acknowledgements
Support was provided in part by Worcester Polytechnic Institute, Green Wisdom Inc., and an Arkansas Biosciences Institute
grant (#262178), DOE Midsouth/Southeast Bioenergy Consortium
(#DE-FG36-08BO88036), Judd Hill Foundation Research Grant
(#159004), National Science Foundation AR EPSCoR P3 Center
(#EPS-1003970) and Arkansas Science & Technology Authority.
We thank the Review Editor Rolf D. Joerger for providing valuable
suggestions.
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