Bioresource Technology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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

Review

Water use and its recycling in microalgae cultivation for biofuel

application
Wasif Farooq a, William I. Suh b, Min S. Park b,c, Ji-Won Yang b,c,⇑
a
Environmental and Energy Technology Program, KAIST, 291 Daehak-ro 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
Advanced Biomass R&D Centre, #2502 Building W1-3, KAIST, 291 Daehak-ro 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
c
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea

h i g h l i g h t s

! Water recycling in microalgae cultivation enhanced the growth of microalgae.

! Presence of extracellular product may enhance bacterial growth.
! Harvesting method affects the water reusability.
! Water reuse resulted in positive energy.
! Reuse of water can reduce nutrient related energy requirement.

a r t i c l e i n f o a b s t r a c t

Article history: Microalgal biofuels are not yet economically viable due to high material and energy costs associated with
Received 6 September 2014 production process. Microalgae cultivation is a water-intensive process compared to other downstream
Received in revised form 26 October 2014 processes for biodiesel production. Various studies found that the production of 1 L of microalgal biodie-
Accepted 28 October 2014
sel requires approximately 3000 L of water. Water recycling in microalgae cultivation is desirable not
Available online xxxx
only to reduce the water demand, but it also improves the economic feasibility of algal biofuels as due
to nutrients and energy savings. This review highlights recently published studies on microalgae water
Keywords:
demand and water recycling in microalgae cultivation. Strategies to reduce water footprint for microalgal
Microalgae cultivation
Water footprint
cultivation, advantages and disadvantages of water recycling, and approaches to mitigate the negative
Water recycling effects of water reuse within the context of water and energy saving are also discussed.
! 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Over the past few decades, the world has observed a gradual
increase in petroleum prices due to political conflicts and depletion
in conventional oil reserves. This has lead on various initiatives to
develop renewable and eco-friendly sources of energy such as bio-
fuels. Biofuels are classified as first-generation, second-generation
and third-generation biofuels. First-generation biofuels i.e. biodie-
sel and bioethanol, are produced from food crops such as rapeseed,
palm, sugar etc. Use of food crops for first generation biofuels ini-
tiated the debate of food vs fuel and land use change for cultivation
of these crops (Chen et al., 2011; Parmar et al., 2011). Second gen-
eration biofuels are obtained from non–food sources such as
⇑ Corresponding author at: Advanced Biomass R&D Centre, #2502 Building W1-3,
KAIST, 291 Daehak-ro 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Repub-
lic of Korea. Tel.: +82 42 350 8862; fax: +82 42 350 8858.
E-mail address: jwyang@kaist.ac.kr (J.-W. Yang).
agricultural residual biomass and waste cooking oil that are con-
verted to bioethanol and biodiesel, respectively. However, the
potential of second-generation biofuels as a source of energy is
limited. Due to limitation associated with first and second-
generation biofuels, a new category of biofuels termed as ‘‘third-
generation biofuel’’ has emerged. Algae-based biofuels belong to
the third generation biofuels (Wigmosta et al., 2011).
Algae belong to a diverse family of organisms ranging from uni-
cellular to multicellular that can thrive on unarable land utilizing
impaired source of water that are unsuitable for agriculture and
municipal use (Menetrez, 2012). They fix the atmospheric CO2 into
their cellular constituents through a biologically process of photo-
synthesis (Menetrez, 2012). Microalgal biomass has been explored
as a potential feedstock for biofuels application for decades. Micro-
algae has higher theoretical energy yield per unit land area com-
pared to terrestrial plants. Microalgae under photosynthetic
growth conditions can achieve 100 times higher theoretical oil pro-
ductivity per hectare compared to conventional energy crops such

http://dx.doi.org/10.1016/j.biortech.2014.10.140
0960-8524/! 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
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2 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx
as soybean. As long as fertilizers are not used, microalgae-based
biofuels production do not compete against human food produc-
tion as has been highlighted with other energy crops such as soy-
bean and corn (Chisti, 2007). Therefore, considerable efforts are
being put to overcome the technical challenges that are associated
with microalga biofuels production, such as high capital and oper-
ating costs of various processes such as cultivation and harvesting,
low photosynthetic conversion efficiency and culture contamina-
tion etc. (Molina Grima et al., 2003; Ugwu et al., 2008), and to
develop a sustainable algal biotechnology for biofuels application.
Large-scale cultivation of microalgae has been carried out for
decades for the production of pharmaceutical and human con-
sumption. Idea to develop microalgae as an energy crop for alter-
native biofuels production is mentioned as early as 1950s, but
has not been considered seriously until petroleum prices crisis in
the 1970s. Due to the concerns of energy security brought on by
the oil prices shock, the US department of energy (DOE) set in
motion a program known as Aquatic Species Program (ASP) from
1978 to 1996 (Murphy and Allen, 2011). The ASP extensively eval-
uated the potential of biodiesel production from microalgae, but
the project eventually abandoned as the crude oil plummeted to
nearly all time low prices in the mid-1990s. However, the recent
spike in the oil prices motivated researchers around the world to
re-examine microalgae as a potential source of biofuels (Zaimes
and Khanna, 2013). Moreover, future fossil fuels consumption
and their negative impacts on environment are expected to
increase. Current world consumption of fossil fuels is estimated
around 4900 Giga liters (GL) per year and 52% of total oil is being
used in the transportation sector. While, current biofuels produc-
tion is around 90 Giga liters per year that is just 1.83% of total fossil
fuels consumption and 3.5% of total fuels supply for the transpor-
tation sector. World oil consumption will be around 6200 GL per
year in 2030, and transportation sector will consume approxi-
mately 56% of total oil produced (Darzins et al., 2010). While, in
the context of the USA alone, a target of 136 " 109 L/yr of renew-
able biofuels production until 2022 is set under Renewable Fuel
standards (RFS) adopted in Energy Independence and Security
Act (EISA). Advanced biofuel must be about 58% of total biofuels
mandate under EISA. Even a 5% contribution from algae as an
advanced biofuel energy crops, will require about 3.975 " 109 of
algal biofuel will be required until 2022. This target will lead to
huge resource requirement such as water, nutrients and land etc.
(Wigmosta et al., 2011). Besides future fuel requirements, reduc-
tion in CO2 emission is required as agreed by nations at the Copen-
hagen Climate Change Summit and a target of 25–40% reduction in
CO2 emission by 2022 and 80–90% by 2050 was set (Stephens et al.,
2010). In order to achieve this goal, use of fossil fuels must be
reduced and renewable sources of biofuels must be explored
(Stephens et al., 2010). Microalgae can consume approximately
2.0 kg of CO2 to produce 1 kg of algae biomass (Kumar et al.,
2011). Microalgae have ability to fix the CO2 during photosynthesis
process. Therefore, it can serve the dual purpose of CO2 mitigation
and biofuels production as well.
Despite many positive characteristics, microalgae cultivation
under nutrients-replete conditions and subsequent oil extraction
from dried biomass have an unfavorable net energy value
(Lardon et al., 2009). A substantial amount of water and fertilizer
requirements are of particular concern for mass production of algal
biofuels, as it can directly compete with direct or indirect use of
these commodities for human consumption (Murphy and Allen,
2011). Life cycle analysis (LCA) can assess the potential impacts
and environmental aspects associated with certain processes and
products. A great deal of studies has been carried out on microal-
gal-based biofuels life cycle analysis (LCA). However, significant
differences in the LCA results are reported (Guieysse et al., 2013;
Liu et al., 2012). This may be due to the complex nature of
Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
http://dx.doi.org/10.1016/j.biortech.2014.10.140
cultivation systems, limited large-scale field data and the low level
of technical advances due to the early stage of microalgal biofuels
research and development (R&D) and industrialization (Guieysse
et al., 2013; Sturm and Lamer, 2011). LCA studies performed on
microalgae biofuels production system also showed diverse results
concerning environmental impacts and energy balance of microal-
gae-based fuels (Clarens et al., 2010; Lardon et al., 2009). Various
investigations have shown that resource demand is expected to
rise when microalgal biofuels production reaches the level that
can make a significant contribution to global demand for transpor-
tation fuel (Pate, 2013). Besides other expensive processes such as
harvesting and drying of algal biomass, microalgae cultivation is
also of major concern in the microalgal biofuels economy on a large
scale. Therefore, a sustainable production pathway must be estab-
lished in order to make algal biofuels industry a reality. Water and
nutrients use and their acquisition shared a major portion of total
operating cost in cultivation system. Water is indispensable for the
survival of microalgal cells, as it acts as a thermal regulator and
provides nutrients delivery (Murphy and Allen, 2011). Large-scale
microalgae biodiesel production has been criticized for the sub-
stantial amount of freshwater usage. It has been reported that in
order to produce 1.0 kg of microalgal biodiesel requires 3726 kg
of water is required (Yang et al., 2011).
The aim of this review paper is to appraise water footprint of
microalgal biodiesel along with water reusability and its recycling
in algal cultivation system. Several environmental and geographic
factors that determine suitable geographical locations for microal-
gal cultivation, with respect to water use, are highlighted. Further-
more, the current paper assessed the energy needs that are
associated with water recycling, new water acquisition and
resource saving such as water and energy as consequence of water
recycling.
2. Water requirement for microalgae cultivation
Microalgal biomass production has been estimated to consume
nearly one metric ton of water per kg of microalgal biomass pro-
duced (Guieysse et al., 2013). Large water consumption is neces-
sary, as microalgae are aquatic species that require a substantial
amount of water for survival and cell proliferation. In addition,
one mole of water dissociates into oxygen and hydrogen per mole
of CO2 consumed during photosynthesis process. Photosynthesis
alone can results in the estimated water loss of nearly 5–10 kg
per 1.0 kg of dry microalgal biomass (Murphy and Allen, 2011;
Williams and Laurens, 2010).
Besides intracellular water, algae are suspended in culture
medium to grow and reproduce in water. Biomass concentration
is about 0.5% of biomass when algae were grown in a photo-biore-
actor (PBR). This means for each kilogram of wet biomass, 200 kg of
non-cellular water is required for growth and support of microal-
gae cultivation (Murphy and Allen, 2011). In the case of open race-
way pond (ORP) where even lower culture density are obtained
due to many factors such as poor light utilization, cell shading
effect and insufficient mixing etc., the culture medium to biomass
ratio is even higher compared to PBR systems. The water require-
ments for a certain product or services are assessed in terms of
water footprint. A concept based on the total volume of fresh water
required to produce a product or services for community or per-
sonal use, at a place where it was produced, is termed as water
footprint (WF). The concept of water footprint (WF) is composed
of three components: green WF, blue WF and gray WF. The green
WF is as defined as the rainwater that is evaporated during the
crop growth period. The blue water footprint is the volume of sur-
face and groundwater consumed during production of a certain
product or service. Consumption includes the volume of
 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx 3

freshwater water evaporated or incorporated into a product or ser- order to keep constant salinity and species control, is assumed to
vice, while gray WF refers to the amount of water required to be same as the evaporation rate (Vasudevan et al., 2012). In order
dilute the pollutant resulting from the production of a product or to control pond crash, contamination and system maintenance,
service to such an extent that quality of natural water system regular pond flushing (two or four times a year) has been sug-
remain above the agreed water quality standards (Gerbens- gested. This results in a discharge of substantial amount of water
Leenes et al., 2009). (Guieysse et al., 2013; Lardon et al., 2009; Murphy and Allen,
The direct water demand is computed as the difference 2011).
between the volume of water required to support the cultivation Various studies have estimated the amount of water needed to
of biofuels crop and annual precipitation (Murphy and Allen, produce an equal amount of microalgae biodiesel. The evaporative
2011; Zaimes and Khanna, 2013) as Eq. (1). water loss alone can be approximately 3 times higher than the
fresh water consumed in irrigated agriculture nationwide, if the
Direct Water demand ¼ Water required to support growth microalgae are cultivation on all the available land in USA. As a
þ Annual precipitation ð1Þ result of this evaporative water loss, on average 1421 L of water
will be required to produce 1 L of algal biodiesel (Wigmosta
Estimation of the total water necessary to support growth of et al., 2011). Water requirement can varied from 3.5 to 3365 L of
culture at optimum condition is based on two key factors. The first water per liter of microalgae biodiesel. Lower end of estimate
one is the amount of water needed to support the growth and sec- assumes effective water capture and recycle (Cooney et al., 2011;
ond is the amount of water that needs to be replaced during Stephenson et al., 2010). Water footprints of microalgae-based bio-
growth as a result of water loss or to compensate for gray water diesel with and without water recycling are given in Table 1. Water
footprint. More precisely, total water input needed for cultivation footprint for microalgae biodiesel is dependent on microalgae
system will be the sum of newly acquired water and water reclaim strain and its lipids productivity. Microalgae strain with higher
along with water loss as shown in Eq. (2) (Murphy and Allen, 2011; lipid productivity will require less cultivation area, so as water,
Zaimes and Khanna, 2013). compare to microalgae strain with low lipid productivity for a unit
V total ; Input ¼ V fill ' Freq þ V evap þ V leakage þ V blowdown þ V photo production of microalgae biodiesel (Yang et al., 2011).
In the majority of LCA analysis microalgal biofuels, water foot-
þ V harvest þ V drying þ V biomass þ V gray water ð2Þ print is calculated based on the biodiesel productivity alone. Mic-
roalgae biomass produced under normal conditions contain
where Vinput = the water volume that needs to be added to the cul-
triacylglycerol (TAG) in the range of 20–30% of dry cell weight.
tivation system annually (m3/m2-yr), Vfill = nominal water required
TAGs are converted to biodiesel. The energy content of TAG based
to fill the cultivation system (m3/m2), Freq empty = the frequency of
biodiesel is 38 MJ/kg, which means that algal biomass contains
emptying the cultivation system for cleaning purpose. Vevap. loss
energy content of 7.6–11.4 MJ/kg as biodiesel energy. Microalgae
(m3/m2-yr) = water loss due to evaporation, Vblowdown (m3/m2-
biomass has significant portion of other energy components such
yr) = the water removed during cultivation to maintain stable salin-
as carbohydrates and proteins besides lipids (Menetrez, 2012),
ity and culture control, and V harvest (m3/m2-yr) = the water
while only the lipids fraction is used for the production of biodiesel
removed during the harvesting process from cultivation pond and
and water footprint calculation. Therefore, it would be more realis-
that is carried over with the biomass. Water loss due to photosyn-
tic to calculate the water demand and footprint based on the total
thesis are very less and can be neglected (Guieysse et al., 2013). The
energy potential of microalgal biomass when algal-based biofuels
gray water footprint can be ignored if the nutrients are perfectly
are compared it with other biofuel feedstocks. Microalgal carbohy-
recycled. The water demand for microalgae production system
drates and proteins can be used for energy applications besides lip-
ranges from 4.59 m3/m2-yr in a tropical region to 6.39 m3/m2-yr
ids from microalgal biomass.
in an arid environment. Water leakage rate from open pond is usu-
Water demand in microalgae cultivation can be met by utiliza-
ally calculated to be 0.0011–0.0036 m3/m2-yr (Guieysse et al.,
tion of wastewater. Utilization of wastewater has been proposed
2013). The major water loss from microalgae cultivation especially
for microalgae cultivation to reduce the fresh water and nutrients
ORPs came from water evaporation. Water evaporation rate can be
load (Farooq et al., 2013b; Rawat et al., 2013). However, the
calculated using different methods such as Pan Evaporation data,
lake evaporation model or use of Penman equation. Most widely
used Penman equation is given as Eq. (3) (Zaimes and Khanna,
2013). Table 1
Water footprint of microalgal biodiesel.
E mass ¼ ðmRn þ c " 6:43 " ð1 þ 0:563 " UmÞ " deÞ=ðkv " ðm þ cÞÞ Water recycle L of water/L of Based on Reference
ð3Þ ratio (R) biodiesel
1 865.88 Chlorella Lardon et al.
where E mass = evaporation rate (mm/day), m = slope of saturation vulgaris (2009)
vapor curve (kPa/K), Rn = net solar radiation (MJ/m2-day), c = Psy- 0 3359.84 Chlorella Yang et al. (2011)
chometric constant (KPaK), Um = wind speed (m/s), de = Vapor pres- vulgaris
sure deficit (kPa), kv = latent heat of evaporation (MJ/kg). 1 526.24 Chlorella Yang et al. (2011)
vulgaris
Water loss due to evaporation based on different estimation 0 1611.84 Chlorella Stephenson et al.
methods are summarized by Guieysse et al., 2013. As expressed vulgaris (2010)
in Eq. (3), water loss due to evaporation is directly related to net 1 3.68 Chlorella Stephenson et al.
available solar radiation and wind velocity. Geographical location vulgaris (2010)
1 1494.08 Theoretical Wigmosta et al.
with higher availability of photo-synthetically active radiation
potential (2011)
(PAR), contribute to higher biomass productivity while at the same 0.9 2744.84 Theoretical Murphy and Allen
time higher water loss is expected due to higher evaporation rate potential (2011)
(Vasudevan et al., 2012). Evaporation losses from the surface of 0 2723.2 Theoretical Cooney et al.
ORPs depends on the local average temperature and relative potential (2011)
0.99 35.328 Theoretical Cooney et al.
humidity as well. Evaporative water loss from an open pond sys- potential (2011)
tem is estimated to be 0.5 cm/day and water blowdown rate, in

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4 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx
availability and location of the wastewater streams is also impor-
tant to consider for its use as a source of water and nutrients for
microalgae cultivation. Co-location of wastewater with suitable
land and abundant sunlight would have a great positive impact
on the microalgal cultivation. Application of LCA tools and the
principles of industrial ecology, where waste streams from one
process are utilized as an input streams for different processes,
may be pivotal in making algal energy systems a practical reality
(Clarens et al., 2010). However, availability of land along with
proximity of wastewater stream, and CO2 source must be evalu-
ated along with GIS system evaluation (Zaimes and Khanna,
2013). Water effluent, after biological treatment of wastewater
with microalgae, can be recycled for further cultivation of microal-
gae in order to reduce the water footprint of microalgal biofuels.
2.1. Effect of geographic locations on water demand and its
accessibility
Cultivation of millions of tons of microalgae per annum requires
vast amount of land and water, which can conflict with land and
water resources for other users ranging from human habitation,
agriculture to the industrial sector. In order to avoid this, mostly
unarable land and wastewater stream must be co-located. Water
footprint of biodiesel varies among different regions. For example,
rapeseed oil based biodiesel has the lowest water footprint in Wes-
tern Europe and highest in Asia, especially India (Gerbens-Leenes
et al., 2009). Similarly, local and seasonal weather will affect the
algal farm management practices and Water footprint as a result.
Local climate has a huge impact on loss of water due to evapo-
ration. The major cause of water loss in the open raceway pond
(ORP) is evaporation. Evaporative loss ranges from 350 mm per
month in Death Valley, California, to 120 mm per month in Landis-
ville in the USA (Wigmosta et al., 2011). Temperature and humidity
at a certain location also plays an important role in water evapora-
tion rate. Water evaporation rate varies from 0.476 m3/m2-yr in a
tropical region to 2.275 m3/m2-yr in a region that has arid climate
conditions (Guieysse et al., 2013). The depth of the water aquifer
for saline and freshwater source is dependent on the geological
location as well (Murphy and Allen, 2011). Water footprint calcu-
lations based on precipitation and evaporation rate alone can over
or underestimate the water demand. Sudden changes in weather
patterns and the occurrence of extreme events such as floods,
drought, monsoons and hurricane will have a significant impact
on water management for microalgal biofuel.
In addition to water footprint, various geographically depen-
dent factors also affect the growth of microalgae. These factors
range from irradiance, temperature, humidity, air speed and cloud
cover (Doucha and Lívanský, 2009; Gao et al., 2007). Each microal-
gae strain has a maximum growth rate at an optimal temperature
and higher or lower temperature beyond the optimum point
affects the growth of microalgae. Temperature below 20 "C and
Table 2
Parameters related to water stress and seasonal variability at the selected location.
Parameter Australia Kenya
Baseline water stressa 3.5 0.7
b
Interannual variability 3.2 3.2
Seasonal variability c 2.0 1.9
Flood occurrence d 2.7 3.8
Drought severitye 1.1 1.6
Legend: 4–5 extremely high (>80%), 3–4 high (40–80%), 2–3 medium to high (20–40%), 1–2 low to medium (10–20%), 0–1 low (<10%).
http://www.wri.org/applications/maps/aqueduct-country-river-basin-rankings/#x=-91.10&y=10.44&l=2&v=home&d=dro&f=0&o=55.
a
Baseline water stress: it is a ratio of total annual water withdrawals to total available annual renewable supply.
b
Variation in water supply between years.
c
Variation in water supply between months of the year.
d
The number of floods recorded from 1985 to 2011.
e
Average length of drought times the dryness of the droughts from 1901 to 2008.
Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
http://dx.doi.org/10.1016/j.biortech.2014.10.140
35 "C on higher end, shows steep decline in productivity of micro-
algae strains used in mass cultivation (Lundquist et al., 2010). Geo-
graphical location with higher availability of photo-synthetically
active radiation (PAR), contribute to higher biomass productivity,
while at the same time higher water loss is expected due to higher
evaporation rate (Vasudevan et al., 2012). Microalgae cultivation in
low temperature regions, below the optimum microalgae cultiva-
tion temperature, will reduce the water evaporation loss and hence
the water consumption, but at the detriment of biomass productiv-
ity (Doucha and Lívanský, 2009; Gao et al., 2007).
Based on the climate data alone, the countries on the Mediter-
ranean Sea coastline area are preferable locations for microalgae
cultivation. In particular, countries in the southern Mediterranean
have warmer climate where temperature does not drop below
15 "C throughout the year. Countries, such as Morocco, Algeria,
Tunisia and Egypt, are particularly attractive due to warm temper-
atures and enormous area of unused desert. While countries like
Libya, Cyprus and Turkey also have large amounts of marginal land
that can be used to grow microalgae (Singh and Gu, 2010).
Although these countries possess ideal conditions for microalgae
biomass production due to availability of unused land, abundant
sunlight and optimum temperature, but these regions have a
severe water availability issue. In Mediterranean regions, the
annual differences between rainfall and evaporation results in a
water loss of 300 mm (Lardon et al., 2009). The use of brackish
and seawater is proposed, but still a large quantity of fresh water
is required to compensate for the evaporative loss and to keep
the water salinity in proper range. In addition, desert regions can
reach extremely high temperatures during summer, which can also
cause culture crash and decrease in overall biomass productivity
(Moody et al., 2014). In general, areas in the temperate regions of
the globe, receive lower light intensity and have fluctuation in sea-
sonal temperatures, while locations along the equator receive
higher solar radiation and have stable year round climate
(Moody et al., 2014). However, other factors such as rain and
cloud-cover over the year will also influence the microalgae biofuel
productivity. For example, region of Manaus in Brazil and Alice
Springs, the third largest town in northern Australia, are both near
the equator, but have different lipid productivity.
The study conducted on the global evaluation of microalgal bio-
fuels potential also concluded that Australia, Cambodia, Brazil,
Egypt, India, Kenya and Saudi Arabia are expected to give maxi-
mum annual lipid yield ranging 24–27 m3/ha-yr. (Moody et al.,
2014). The estimation was based on the assumption that water,
nutrients and CO2 are not limited. However, water availability
throughout the year is not identical in different parts of the world.
In the Mediterranean and Middle East, many states are already
under severe water stress. Water risk indicator such as water
stress, seasonal variability, flood occurrence and drought severity
of several countries are given in Table 2. Most of the states
that have favorable climate conditions to achieve high lipid
Brazil Colombia Egypt Ethiopia India Saudi Arabia
0.9 0.3 1.3 0.6 3.6 5.0
1.7 1.3 1.4 1.8 1.7 3.5
2.5 1.5 2.5 3.3 4.2 1.6
3.1 3.7 2.5 3.2 3.9 1.2
1.1 1.6 3.4 1.4 1.7 2.1
 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx
productivity are already under serious water stress situation (WRI,
2014), and further usage of water for microalgae cultivation will
result in the fuel vs water debate. Furthermore, data in Table 2
showed that environmental and economic price of one ton of water
in a desert or water stressed area is not equivalent to those in the
tropical regions where surface water is abundant. In order to
account for these critical factors, a different unit known as ‘‘num-
ber of years of local rainfall required for unit production of energy’’
is suggested as an alternative to traditional water footprint
(Guieysse et al., 2013). All these factors are compelling reasons to
maximize the water use in microalgae cultivation system with
effective reclaim and recycling system. As a result, various studies
have reported the water recycling and reuse in microalgae
cultivation.
2.2. Reuse of water in microalgal cultivation
LCA studies performed on microalgae cultivation systems
(Clarens et al., 2010; Yang et al., 2011), concluded that economics
of microalgae-based biodiesel will be improved significantly by
reducing water and nutrients demand. This can be met by contin-
uous reuse of culture medium and using impaired water sources
such as wastewater. However, 100% water recycling is impossible
even under perfect operation. Significant amount of water is lost
due to water evaporation and during harvesting and drying of mic-
roalgae biomass (Guieysse et al., 2013). Since majority of the water
footprint comes from the cultivation system, it is necessary to
recover and recycle the maximum amount of used medium for fur-
ther cultivation. In addition, a large quantity of unused nutrients
loss is expected if water after the harvesting process is not recircu-
lated into the cultivation system. This will also contribute to the
gray water footprint. Water recycling after biomass harvesting is
expected to reduce the water requirement and nutrients demand,
which is essential for long-term sustainability of microalgal
biofuels.
2.2.1. Effect of water recycling on the quality and quantity of biomass
There have been debates on the potential advantages and disad-
vantages of the reuse/recycling of culture medium for subsequent
cultivation of microalgae. Contrary to the results from an earlier
study (Rodolfi et al., 2003), recent studies solidly demonstrated
the possibility and benefits of water recycling along with the asso-
ciated, and challenges for the long term reuse of culture medium
(Fon Sing et al., 2014).
Microalgae Tetraselmis MUR 233 was cultivated in a raceway
pond for a period of five months using both fresh and recycled
water under semi-continuous cultivation mode using electro-floc-
culation based harvesting. Tetraselmis MUR 233 was able to grow
continuously in recycled water with minimal freshwater input
without any decline. However, the water salinity increased from
5.5% to 12% (w/v) of NaCl due to evaporation of water. Tetraselmis
MUR 233 growth using recycled water resulted in 48–160%
increase in ash free dry weight (AFDW) of biomass daily compared
to control sample. Peak productivity of 37.5 ± 3.1 g AFDW m( 2 d( 1
was achieved when the salinity of recycled water was reduced to
5% NaCl. High growth was expected as a result of mixotrophic
growth conditions (Fon Sing et al., 2014).
Similar results have been reported for different microalgae dur-
ing use of recycled water cultivation. Water recycling after nano-
chitosan based harvesting of microalgae Nannochloropsis sp.
growth in the recycled water was 7% higher compared to control
medium after nano-chitosan based harvesting. The growth
enhancement was attributed to the use of acetic acid that was used
for pH adjustment before cultivation (Farid et al., 2013). Growth of
microalgae Scenedesmus sp. was enhanced during use of recycled
water obtained after bioflocculant-based microalgae harvesting.
Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
http://dx.doi.org/10.1016/j.biortech.2014.10.140
5
However, use of alum as harvesting agent deteriorates the recycle
water quality. As a result, the use of recycled water reduce the mic-
roalgae growth rate due to toxic effect of residual Al in recycled
water (Kim et al., 2011). While, microalgae Nannochloropsis salina
was grown successfully in recycled water after aluminum nitrate
sulfate based flocculant (Rwehumbiza et al., 2012).
Growth of microalgae, Nannochloropsis oceanica was enhanced
during cultivation in water effluent coming from electro-floccula-
tion based harvesting stage (Kim et al., 2014). Cells growth was
enhanced by nearly 16% in the recycled water compared with the
control medium. Enhanced growth is attributed to the presence
of extracellular polysaccharides released by microalgae into the
water during its growth. Released carbohydrates can act as addi-
tional carbon sources that facilitates superior algal growth in recy-
cled water (Kim et al., 2014). It has been reported that the release
of extracellular organic molecules in water during microalgae
growth can enhance the growth of microalgae (Becker, 1994). Mic-
roalgal cells exhibited higher quantum yield when grown in recy-
cled water compared to that of control medium (Kim et al., 2014).
Continuous as well as batch cultivation of microalgae Nanno-
chloropsis gaditana was carried out using recirculated sterilized
supernatant (González-López et al., 2013). Reuse of water during
cultivation has no negative impact on biomass quality.
(González-López et al., 2013). Growth of microalgae, Chlorella
vulgaris, was enhanced when the processed water from the hydro-
thermal carbonization process was used (Zhu et al., 2013). Impact
of water recycling on growth of microalgae C. vulgaris was also
investigated in terms of its biomass productivity, quality of bio-
mass and accumulation of extracellular metabolites in the culture
medium during growth (Hadj-Romdhane et al., 2013). Results
showed that growth of C. vulgaris was not inhibited even after
63 days of water recycling, and biomass productivity remained
consistent at around 0.55 kg/m3 day1. Composition of extracellular
organic materials released by microalgae in the water mainly con-
sisted of biopolymers (BP) with sizes above 40 kDa (probably poly-
saccharides), along with nitrogen-rich small organic molecules
(SOM) having molecular size ranging from 1 to 3 kDa. During the
reuse of recycled water, concentration of biopolymers was found
to increase initially, and then decrease. The subsequent decrease
was most likely due to the growth of bacteria (Hadj-Romdhane
et al., 2013). This work highlighted the issue of unused nutrients
during microalgae cultivation and chances of contamination due
to extracellular products released by microalgae growth its growth.
The accumulation of unused cations and anions (Na, Ca and Cl),
that was not assimilated by microalgae during its growth, resulting
in increased water salinity. It was proposed that an effective for-
mulation of fertilizer and monitoring strategy to assess the accu-
mulation of nutrient is essential for economic and ecologically
viable cultivation of microalgae (Hadj-Romdhane et al., 2012).
Growth of marine microalgae Nannochloropsis sp. was
decreased during the reuse of recycled water (Rodolfi et al.,
2003). The growth rate of microalgae decreased compared to the
control even after the essential growth nutrients were replenished.
It might be due to the toxicity of extracellular products released
into water during microalgae cultivation. Presence of particulate
matter also expected to affect the growth of the microalgae. The
particulate materials in the recycled water were composed of
mainly ‘‘aging’’ cell walls that can make large cell aggregates. After
removal of particulate matter, biomass productivity was improved
by 21% (Rodolfi et al., 2003). Microalgae C. vulgaris was grown suc-
cessfully in recycled water when water was supplemented with
essential nutrients after organoclay based harvesting (Farooq
et al., 2013a).
Our recent study also draws similar conclusions with C. vulgaris
at laboratory scale. Our study further shed lights on the role of
residual components toward growth of microalgae during water
6 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx

recycling, influence of the harvesting methods on the quality of
recycled water, and quality of biomass and biodiesel. Both centri-
fugation and flocculation with FeCl3 are equally effective in terms
of achieving high harvesting efficiency without having any delete-
rious effects on recycling of the used medium for subsequent cul-
tivation. However, the residual alum showed inhibitory effect on
growth of microalgae C. vulgaris. The presence of extracellular sub-
stances such as carbohydrate, proteins, and residual flocculant
such as ferric ions in the recycled media can explain the positive
effects of water recycling. Residual ferric ions that are present in
both harvested biomass and biodiesel was easily removed through
a simple change of pH or a washing step with water. Our results
suggest that the use of recycled water for microalgal cultivation
is possible. However, the choice of harvesting methods must be
made carefully when the recycling of culture medium is consid-
ered for microalgal cultivation. The results of studies conducted
on water recycling for microalgal cultivation are summarized in
Table 3.
2.2.2. Effect of harvesting method on the quality of recycled water
There is a concern about the use of chemical flocculants for mic-
roalgae harvesting. Metal-based flocculants can contaminate the
harvested biomass, and any residual flocculant in the residual
water can deteriorate the quality of recycled water for its reuse
in cultivation system, in addition to posing environmental threat
(Molina Grima et al., 2003). Aluminum based flocculants have been
reported to inhibit the growth of microalgae during the course of
water reuse (Kim et al., 2011). Metal flocculants can be replaced
by using harvesting methods that are based on pH change. Such
methods can harvest microalgae cell without any chemical con-
tamination. However, the recycled water obtained after pH-based
microalgal harvesting requires neutralization and supplementation
of depleted essential nutrients in order to support the growth of
microalgae. High pH was used to harvest several freshwater and
marine microalgae, and remaining water after harvesting was neu-
tralized using HNO3 (Vandamme et al., 2012; Wu et al., 2012).
Bioflocculant based microalgal harvesting is also an environ-
mentally friendly and chemical-free approach. In one study, Scene-
desmus sp. was harvested using both inorganic flocculant and
bioflocculant produced by P. polymyxa AM49 (Kim et al., 2011).
During reuse of recycled water, high growth and biomass yield
was observed. Harvesting of microalgae based on change in pH of
culture and bio-flocculant based harvesting method can support
the reusability of the water, while avoiding the risk of chemical
contamination in the water and the desired final products. During
water recycling, all microalgal species showed similar biomass
productivity under the recycled medium compared with the fresh
medium, showing successful recycling of medium for microalgal
cultivation (Liu et al., 2013). Similarly, microalgae flocculation
can be induced at lower pH without use of any additional floccu-
lants (Liu et al., 2013).These finding suggested that while making
a choice among energy intensive and cheap ways of microalgae
harvesting, their potential impact on water recycling and quality
of final product must be considered..
2.2.3. Pretreatment of recycled water
The possibility of water recycling in microalgae cultivation
depends largely on the suitability of the water for the subsequent
cultivation. The extracellular metabolites released by microalgae in
water can limit the reuse of water. These secreted metabolites can
potentially carry over and accumulates during water recycling.
Gradual accumulation of toxic compounds released by microalgae
can also cause problem during water recycling (Cooney et al.,
2011) and lead to deterioration of quality of the recycled water.
These compounds may include, proteins, carbohydrates, cell wall
debris, contaminating organisms, dissolved organic compounds
and other potential growth inhibiting chemicals released from
the cells, or accumulated heavy metals. Accumulation of water sol-
uble extracellular products released from microalgae during the
reuse of water not only can have inhibitory effects on the growth
of microalgae (Zhang et al., 2013), but can also lead to bacterial
activity and pond crash (Zhang et al., 2010). The extracellular prod-
ucts could potentially be a cause and source of bacterial growth
even in the controlled cultivation environment such as closed
PBR system (Hulatt and Thomas, 2010).
Therefore, pretreatment of recycled water before returning it to
the cultivation system might be essential. One possible solution is
the use of membrane technology to purify the water from patho-
genic microbes and extracellular products. However, the price of
membrane filtration can negatively impact the overall economics
of microalgal biofuels (Guieysse et al., 2013). Studies on water

Table 3
Studies on water recycling in microalgae cultivation.

Strain Cultivation Harvesting Mode and Impact of water reuse compared to References
system technique No. of control
recycle
Tetraselmis sp. ORP Electro flocculation Continuous 48–160% more AFDW Fon Sing et al.
(5 months) (2014)
Nannochloropsis sp. PBR (120 L) Centrifugation Batch (2) Biomass was decreased 50–60% of Rodolfi et al.
control (2003)
Tetraselmis sp. ORP Electro flocculation Continuous Recycled water have higher bacterial Erkelens et al.
(120000 L) and Centrifugation (1) load (2014)
Nannochloropsis oceanica ORP Electrolytic Batch (1) 16% higher growth compared to Kim et al.
(10000 L) harvesting control (2014)
Nannochloropsis salina PBR Alum (2–16 ppm) Batch (1) Reuse water support the growth, no Rwehumbiza
alum toxicity et al. (2012)
Chlorella vulgaris PBR (1L) Centrifugation Continuous Biomass productivity remain stable Hadj-
(63 days) Romdhane
et al. (2013)
Nannochloropsis sp. PBR (2 L) Nano chitosan Batch (1) Recycled water give 7% high Farid et al.
productivity (2013)
Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp., PBR (Lab pH induced Batch (1) Give same biomass productivity (Wu et al.
Nannochloropsis oculata, Phaeodactylum tricornutum scale) flocculation (high (2012)
pH)
Scenedesmus sp. Lab scale Bioflocculant and Batch Growth was enhanced after use of Kim et al.
alum bioflocculant but alum showed (2011)
toxicity

ORP = open raceway pond, PBR = photobioreactor, AFDW = ash free dry weight.

Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
http://dx.doi.org/10.1016/j.biortech.2014.10.140
 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx
recycling in microalgae cultivation have adopted different methods
to treat the recycled water before its reuse in cultivation system.
Water was allowed to settle overnight to remove any floccu-
lated cells before it was pumped back into cultivation pond (Fon
Sing et al., 2014). While, high-speed centrifugation (10,000"g)
was used to remove the particulate matter from the supernatant
before it was recycled for cultivation. Water recycling after
removal of particulate matter gives better growth compared to
untreated one (Rodolfi et al., 2003). It has been reported that water
effluent after centrifugation (4200 rpm) based harvesting has a sig-
nificantly lower bacterial count compared to the water obtained
after electroflocculation based harvesting (Erkelens et al., 2014).
Water recycling after microalgae harvesting using high-speed
centrifugation (17,600"g) enhanced the microalgae growth.
Harvesting at such a high speed can eliminate bacteria and other
inhibitory particulate matter as well (Hadj-Romdhane et al.,
2013). During high-speed centrifugation (10,000–17,600"g) as a
harvesting or pretreatment step, a significant amount of particu-
late matter and bacterial load in recycled water was decreased.
Several sterilization techniques including filtration, ozonation,
chlorination, hydrogen peroxide and heating were tested to evalu-
ate their effectiveness with respect to sterilization of water for
microalgae cultivation. Ozonation was more effective to reduce
the bacterial load from the water. The bacterial load in the recycled
water after ozonation was 1000 and 10-fold lower than the water
after harvesting and initial filtration of water after harvesting,
respectively (González-López et al., 2013). Continuous as well as
batch cultivation of microalgae N. gaditana was carried out using
this recirculated sterilized supernatant (González-López et al.,
2013). However, the application of these sterilization approaches
may not be practical to commercial scale microalgal ponds.
Salinity of the recycled water is expected to increase in marine
culture due to evaporative water loss. Concentration of various
extracellular products can also increase under higher salinity in
case of marine algae (Mishra and Jha, 2009). Reverse osmosis has
been proposed as a pretreatment process to control the salinity.
However, it is a very expensive process on energy scales and man-
agement of accumulated salt after reverse osmosis is another issue.
In this case, the use of freshwater for dilution of high salt concen-
tration is the only solution, although this will result in the increase
of the freshwater requirement. Difficulty in adjusting salinity in
recycled medium adds a significant cost to the recycling of water
for marine species. Moreover, water with excessive extracellular
products and unused nutrient must be treated before its discharge
into sea. Otherwise, this may result in environmental problems for
aquatic life. Based on all these factors, it has been concluded that
use of saline water can be an expensive alternative (Pate et al.,
2011).
Pretreatment of recycled water have been carried out in small
scales, but it is almost impractical to use these pretreatment meth-
ods for water recycling in commercial-scale microalgae farms.
These results showed that besides cost of microalgae harvesting
Table 4
Percentage of energy required to compensate for direct and total energy used water management without intermediate storage.
% of the biomass energy required to compensate for direct energy used during water management acquisition
Water recycle (%) Energy from Biodiesel (%)
R=0 37–39
R = 90 13–14
% of the biomass energy required to compensate for total energy used during water management acquisition
Water recycle (%) Energy from Biodiesel (%)
R=0 96
R = 90 31
Total energy = direct energy + upstream energy
Upstream energy = 2.57 of direct energy in case of electricity
Please cite this article in press as: Farooq, W., et al. Water use and its recycling in microalgae cultivation for biofuel application. Bioresour. Technol. (2014),
http://dx.doi.org/10.1016/j.biortech.2014.10.140
7
methods, their potential positive and negative impacts on water
quality after harvesting must be evaluated. Moreover, cost and
economics of integrating a pretreatment step after primary har-
vesting to improve the recycled water quality must be assessed
along with overall cost of microalgal-based biofuels. A desirable
harvesting method, in context of water recycling, quality of har-
vested algal biomass and biofuel price, must have energy lower
input along with its ability to remove the growth inhibiting ele-
ments from water before its recycling in microalgae cultivation.
2.3. The energy savings by water recycle
In order to replace the 25% of the USA transportation fuels with
microalgal-based biodiesel, 1.73 " 1012 kg/yr. of algal biomass is
required. The quantity of water required meeting this demand of
microalgal biomass is equal to 1100 " 109 m3/yr. (Murphy and
Allen, 2011). The management and acquisition of this huge quantity
of water will consume a substantial amount of energy. The amount
of energy required to meet the water demand depends on the type
of water used for cultivation. Both fresh and saline water can be
used depending on the microalgal strain and location of cultivation.
In case of fresh water, the energy required is 1.3 MJ/m3 based on
the USA conditions and acquisition (Murphy and Allen, 2011). The
energy demand to supply the underground saline water to the cul-
tivation system is dependent on the depth of aquifer. Theoretical
energy required to pump water from underground sources is
estimated to be additional 0.02 MJ/m3 of water per meter of depth
(Murphy and Allen, 2011). Depth of water aquifers in USA continent
varies from 75 to 380 m. At an average depth of 180 m, additional
3.6 MJ/m3 of energy will be required to supply the underground
saline water. Due to the additional energy requirements of using
underground saline water sources, utilization of available surface
water appears to be the best choice for microalgae cultivation.
Therefore, energy required to acquire the water source also impact
the overall microalgal economics.
The outcomes of water recycling in microalgae cultivation have
been discussed within the context of biomass productivity, pond
management, and with respect to price of water commodity. How-
ever, it is also necessary to estimate the net energy gain or loss that
is associated with the reuse of water. Water recycling requires
energy to pump the reclaimed water back into cultivation system
from sedimentation pond after primary settling. Based on a race-
way pond with area of 10,000 m3 and 0.3 m water depth requires
3000 m3 of water as described by Murphy and Allen, 2011. Based
on daily harvesting half the water, 90% (R = 0.9) of water is avail-
able for recycling to cultivation system as described by (Murphy
and Allen, 2011). Energy required to recycle the harvested water
(R = 0.9) and acquire the fresh water to compensate for the water
losses due to evaporation, blowdown and leakage, is about 13–15%
of total energy obtained from microalgal biomass as biodiesel.
Water was pumped directly to cultivation system after harvesting.
Energy requirement is further increased to 31% if upstream energy
% of Energy from CH4 via Anaerobic digestion of LEA (%)
57
19.22
% of Energy from CH4 via Anaerobic digestion of LEA (%)
148
49.41
8 W. Farooq et al. / Bioresource Technology xxx (2014) xxx–xxx
was also accounted. However, without water recycling (R = 0), a
huge amount of energy will be required to compensate for the
energy demand to acquire new water for cultivation system. With-
out water recycling (R = 0), approximately 40% of biodiesel energy
produced from the microalgae biomass will be required to supply
the water to cultivation system. While considering upstream
energy, 96% of the energy produced as biodiesel from microalgal
biomass will be used to acquire fresh water in USA context, as
given in Table 4. Energy requirement for water recycling can be
met by energy produced from the lipid-extracted microalgae
(LEA). Microalgae lipids extracted biomass (LEA) contains a signif-
icant amount of energy, because it has carbohydrates, proteins, and
some residual oil from the incomplete extraction.
Our calculations suggested that water recycling is important
not only to reduce the water footprint, but also to minimize the
energy for microalgal biofuels production. Water recycling not only
reduces the water demand for a unit production of biodiesel, but it
can also save the unused nutrients remaining in the water after
microalgae cultivation. It has been reported that the use of nitro-
gen and phosphorus for cultivation of microalgae C. vulgaris was
reduced to 55% when 100% water was recycled (Yang et al.,
2011). Energy required to produce 1.0 g of nitrogen and phospho-
rus fertilizer is 60 kJ/g and 15 kJ/g, respectively (Menger-Krug
et al., 2012). As a result, of 100% water recycling, 45% of energy
consumption connected to nitrogen and phosphorus production
can be saved per kg of biodiesel produced from microalgae
C. vulgaris.
3. Conclusion
Water footprint of microalgal biofuels is highly dependent on
local environmental and geographical conditions. Water recycling
in microalgae cultivation can be an effective way to reduce the
overall water consumption. Quality of recycled water depends on
harvesting methods. Release of extracellular products by microal-
gae may pose a serious challenge in water recycling. Long-term
studies are required to elucidate the challenges of water recycling
for microalgal cultivation on large scale. Effects of water recycling
on energy balance, biomass production cost, and long-term
sustainability of water and nutrients resources must be carefully
considered to make the microalgal biofuels industry a reality.
Acknowledgement
The Advanced Biomass R&D Center (ABC) of Korea Grant funded
by the Ministry of Science, ICT and Future Planning (ABC-2010-
0029728) supported this work.
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