Bioresource Technology 302 (2020) 122817

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

Review

Resource recovery from wastewaters using microalgae-based approaches: A T

circular bioeconomy perspective
Dillirani Nagarajana,b, Duu-Jong Leeb,c, Chun-Yen Chend, Jo-Shu Changa,e,f,
⁎

a
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
b
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan
c
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
d
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
e
Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung, Taiwan
f
Center for Nanotechnology, Tunghai University, Taichung, Taiwan

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: The basic concepts of circular bioeconomy are reduce, reuse and recycle. Recovery of recyclable nutrients from
Circular bioeconomy secondary sources could play a key role in meeting the increased demands of the growing population.
Microalgae Wastewaters of different origin are rich in energy and nutrients sources that can be recovered and reused in a
Wastewater circular bioeconomy perspective. Microalgae can effectively utilize wastewater nutrients for growth and biomass
Bioremediation
production. Integration of wastewater treatment and microalgal cultivation improves the environmental impacts
Phyco-remediation
of the currently used wastewater treatment methods. This review provides comprehensive information on the
potential of using microalgae for the recovery of carbon, nitrogen, phosphorus and other micronutrients from
wastewaters. Major factors influencing large scale microalgal wastewater treatment are discussed and future
research perspectives are proposed to foster the future development in this area.

1. Introduction innovative biological processes and principles to sustainably provide

goods and services across all economic sectors”. In essence, it is the
Bioeconomy, as defined at the Global Bioeconomy Summit, is “the much needed and highly beneficial transition from finite resource
knowledge based production and utilization of biological resources, (fossil fuels) based economy to a sustainable bio-based economy. The

⁎
Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).

https://doi.org/10.1016/j.biortech.2020.122817
Received 21 December 2019; Received in revised form 10 January 2020; Accepted 11 January 2020
Available online 20 January 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
D. Nagarajan, et al.
basis for raw materials or key intermediates in many industries need to
be of biological origin, thus being sustainable and green. Agriculture
based biomass or virgin biomass derived from the vast forest resources
can be utilized as industrial feedstock. Conventionally, many industries
like the paper/pulp industry, food industry, breweries and a major part
of the pharmaceutical industry (vaccines and pre-, pro-biotics) are bio-
based. An extension of the application of these bio-based resources to
other industries that support economic growth, such as the fuel and
energy sector would be more beneficial because of the higher product
quality, a better lifestyle (amended by pollution free air, water, and
soil) that successfully meets the demands of an ever growing popula-
tion. Bioresource-based economy is advantageous for the following
reasons: reduction in greenhouse gas emissions, sustainable resources,
better environmental protection (from non-degradable and persistent
pollutants) and devoid of secondary pollution. Circular economy, as the
name indicates, is the better use of resources in a closed looped manner
as opposed to the linear manner of fossil fuel based economy. Circular
bioeconomy oversees the better and complete utilization of the pro-
posed biomass maintaining sustainability and ecological balance, while
also promoting the major goals of a circular bioeconomy: reduce, reuse
and recycle. In this review, the “reduce” and reuse” part of the circular
bioeconomy will be discussed in detail in the context of the high nu-
trient loads of various wastewaters and the use of microalgae for
bioremediation of wastewaters in a circular bioeconomy concept.
Wastewater is generated in many quarters of the society, during
domestic and industrial activities. In Tehran, a total of
186.06 ± 7.85 L/day/capita domestic wastewater was generated
(Mesdaghinia et al., 2015). Conventional wastewater treatment
methods generate a highly concentrated municipal sludge, which needs
to be disposed of effectively without leading to secondary pollution. In
the United States of America, 6.5 million metric tons of dry municipal
sludge is generated annually after wastewater treatment (Venkatesan
et al., 2015). Disposal of sludge by anaerobic digestion is the main
beneficial route, but the Environmental Protection Agency (EPA, USA)
indicates that 14.2 and 5.0 MMT CO2-equivalent of CH4 and N2O were
released during sludge digestion in wastewater treatment plants in
2017 (U.S. EPA (2019) Inventory of U.S. Greenhouse Gas Emissions and
Sinks 1990–2017). Annual production of wastewaters from con-
centrated livestock feeding operations was approximately 2170 million
tons in 2010 (Pan et al., 2011). Animal manure in wastewaters in-
creases the total and ammonia nitrogen content of the wastewater, in
addition to the organic matter present. Pharmaceutical and other metal
industry wastewaters contains organic micro pollutants and heavy
metals which needs to be removed prior to water reclamation for reuse
or safe environmental discharge (Cai et al., 2013). While wastewater is
regarded as a potential problem in many facets, the energy content of
wastewater related to the chemical oxygen demand was estimated to be
6.3 kJ/L. A precise measurement using freeze drying of the samples for
Fig. 1. Microalgae based wastewater treatment and circular bioeconomy in resource efficiency.
2
Bioresource Technology 302 (2020) 122817
minimizing volatiles/organic matter loss determined the energy content
of mixed wastewater sample and domestic wastewater to be 16.8 kJ/L
and 7.6 kJ/L, respectively (Heidrich et al., 2011). In this aspect, was-
tewater is an abundant source of energy to be recovered and reused, a
very vital circular bioeconomy perception.
Furthermore, wastewaters are rich in organic/inorganic forms of
carbon, nitrogen, phosphorus. Anthropogenic interventions in the
global biogeochemical cycles has resulted in a drastic one-way mobi-
lization of these resources into the atmosphere and the environment
(Elser & Bennett, 2011). Even though nitrogen is available in abun-
dance in the atmosphere, the production of fertilizers for plant nutrition
is a highly energy consuming process. Total energy consumption for the
production of ammonia, phosphate and potash fertilizers by chemical
synthesis was estimated to be 78, 230 kJ/Kg, 17,500 kJ/Kg and
13,800 kJ/Kg, respectively (Gellings & Parmenter, 2004). Also, the
production of ammonia by the Haber Bosch process is responsible for
1–2% of global energy consumptions and 1.44% of global CO2 emis-
sions (Kyriakou et al., 2020). In microalgal cultivation, this indirectly
contributes to about 40% of the total energy input (Peccia et al., 2013).
Phosphorus, on the other hand, is a finite resource and the current re-
serves have a high possibility of depletion within 100 years, with def-
icits starting approximately from the year 2070 due to increased de-
mand. This might also result in high prices and reliance on single point
sources, giving them monopoly over the market (Cooper et al., 2011).
Studies indicate that extraction of phosphorus from phosphate rock
reserves and application as crop fertilizer has increased the P reserves –
land – water flow, resulting in an enhanced soil P accumulation of
6.9 ± 3.3 terra gram-P per year (Yuan et al., 2018). In Brazil, the
major cash crop sugarcane is a high P demanding crop and requires
about 35 Kg P/hectare/year (Soltangheisi et al., 2019). This increased P
demand and the impending P depletion scare has prompted the Brazil
government to adapt a P conservation strategy by realigning P inputs,
reduce P losses by prohibiting pre-harvest land burns, reduce and re-
cycle P from secondary resources (such as wastewater) and redesign
sugarcane farming practices. If practiced, this could save about 305 Gg
of P and reduce P requirements by 63% by the year 2050 (Soltangheisi
et al., 2019). Thus, wastewater P is considered as a renewable source of
P, even though certain authors debate the depletion of P reserves
(Cordell and White, 2011). Instead of releasing the N and P rich was-
tewater into coastal and inland waters increasing eutrophication risk,
utilization of the same microalgal growth for beneficial purposes in
microalgae based wastewater treatment could result in multiple bene-
fits like nutrient recovery, beneficial biomass production, enhanced
water footprint of the wastewater treatment process, water reclamation
for reuse and maintenance of ecological balance in aquatic systems. The
process of nutrient recovery from wastewater and their benefits are
illustrated in Fig. 1. In this review, the nutrient and pollutant removal
capabilities of microalgae will be discussed, alongside the performance
D. Nagarajan, et al.
of microalgal bioremediation of different wastewaters.
2. Microalgae and wastewater treatment
Microalgae are prokaryotic/eukaryotic organisms capable of carbon
fixation via photosynthesis. The major advantage of microalgae as a
third generation biofuel feedstock lies in the fact that the attainable
photosynthetic efficiency in microalgal cultures are about 8.3% while
cultured in the presence of CO2, while the maximum attainable pho-
tosynthetic efficiency of C3 cannot exceed 2.4% annually (Chisti,
2013). This, alongside the vast phylogenetic and physiological diversity
of microalgae, brings out the unique nature of microalgae in being a
potential biomass feedstock. The diversity of microalgae enables dif-
ferent species to grow in different cultivation conditions such as high
CO2, high ammonia, acidic/basic pH, higher/lower temperature, varied
salinity and so on and so forth. When it comes to biofuel production,
cost efficiency in the key, and microalgae based biofuels are not com-
petitive to fossil fuels mainly due to the high costs associated with
microalgal cultivation, harvesting and dewatering. Every Kg of algal
biomass produced require 367 g of carbon, 88 g of nitrogen, 2 g of
phosphorus and 0.001–0.11 m3 of waster based on the process re-
quirements. The water required can be higher if the water required for
fertilizer and energy production are taken into account (Mayers et al.,
2016). If the carbon source is gaseous CO2, 1.5–2.2 Kg of CO2 can be
fixed by the production of 1 Kg of algal biomass, assuming the carbon
content of the biomass is 50% (Mayers et al., 2016). Wastewaters are
typically rich in nutrients required for microalgal cultivation - nitrogen
and phosphorus. They also contain carbon in many forms, and certain
microalgae can metabolize this carbon in a mixotrophic/heterotrophic
mode for biomass production. Production costs for 1 Kg of Scenedesmus
obliquus biomass by outdoor cultivation using nutrient medium and
sunlight, indoor cultivation using nutrient medium and artificial illu-
mination and finally outdoor cultivation with wastewater nutrients and
sunlight were estimated to be 83.19€, 1188.99€ and 31.61–33.38€,
respectively (Gouveia et al., 2016).
2.1. Advantages of microalgae based wastewater treatment
Microalgal cultivation in wastewater provides simultaneous bio-
mass production and wastewater bioremediation. The advantages of
microalgae based wastewater treatment are illustrated in Fig. 2 and are
Fig. 2. Advantages of microalgae-based wastewater treatment.
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Bioresource Technology 302 (2020) 122817
discussed below:
(1) Microalgae based wastewater treatment systems can be successfully
integrated with existing wastewater systems for effective algae
based biofuels production based on life cycle analysis (Roostaei and
Zhang, 2017).
(2) The costs associated with microalgae based wastewater treatment
are lower compared to the conventional treatment processes.
(3) The energy requirements of microalgae based wastewater are low
compared to the conventional activated sludge process due to the
high levels of oxygenation required for bacterial metabolism.
Energy required for the treatment of wastewater using conventional
technologies in Spain was estimated to be 0.5 kWh/m3 wastewater,
while using microalgae based methods its can be reduced to
0.2 kWh/m3 wastewater (Acién Fernández et al., 2018). Microalgae
utilize sunlight for the assimilation of wastewater nutrients and
photosynthetic conversion of carbon to biomass.
(4) The nutrients are removed by dissimilatory mechanisms in the
conventional methods finally dissipating components like N and C
to the atmosphere, while microalgae based bioremediation removes
nutrients by assimilatory mechanisms and thus the nutrients can be
recovered in the biomass. The emissions associated with the treat-
ment of 500 Mm3 of wastewater for CO2, N and P were 1000 kt,
25 kt and 5 kt per year, respectively (FCC Aqualia, Spain). If mi-
croalgae based methods are to be used, the same amount of treated
wastewater would produce about 500 kt of microalgal biomass per
year, with the nutrients assimilated in the biomass rather than
dissipated to the atmosphere (Acién Fernández et al., 2018). Re-
covery of nutrients and resource efficiency is a key circular bioec-
onomy concept.
(5) The nutrient removal efficiency of microalgae is higher for the le-
vels of nitrogen and phosphorus present in many wastewaters.
Wastewaters from animal husbandries (cattle, swine, and poultry)
are rich in ammonia, but certain microalgae shows high tolerance
towards ammonia rich wastewaters as well (Nagarajan et al., 2019).
(6) As seen in points 2 and 3, the GHG emissions in microalgae based
wastewater treatment is low compared to conventional methods,
based on the energy requirements and the carbon sequestration
potential of microalgae. Microalgae can also sequester carbon from
point sources, reducing GHG emissions.
(7) Conventional wastewater treatment by activated sludge based
D. Nagarajan, et al.
method generates a sludge, which is also nutrient rich and needs to
be disposed off (Venkatesan et al., 2015). Anaerobic digestion if
sludge results in the secondary wastewater called centrate, which is
also treated as a wastewater due to its high nutrients content. The
products of a microalgae wastewater treatment are a beneficial
biomass and a treated effluent very low in nutrient content.
(8) Even though wastewater microalgae cannot be used for human
consumption, they are a great sustainable alternative for animal
and aquaculture feed (Van Den Hende et al., 2016). Based on the
biochemical composition, they can be used for the production of
various biofuels. Biomass residues obtained after the recovery of
proteins or lipid or carbohydrates, also known as spent microalgal
biomass, can further be subjected to energy recover or other ap-
plications (Guedes et al., 2019).
2.2. Selection of the suitable microalgal strain for wastewater treatment
Microalgae based wastewater treatment process requires a suitable
microalgal strain that could tolerate the extreme conditions present in
the wastewater. The COD/BOD levels, N and P present in anaerobic
digestates and agro-industrial wastewaters are higher in the order of
3000–16,000 mg/L, 30–9000 mg/L and 10–500 mg/L, respectively
(Acién Fernández et al., 2018). Even though the conservative average
estimate of the COD/BOD of agro-industrial wastewater is < 20,
000 mg/L, very high COD levels have been reported. The COD level of
raw and anaerobically digested cattle manure was 38,230 mg/L and
23,760 mg/L, respectively (Wang et al., 2010). However, the COD/
BOD, N and P levels of domestic/urban wastewater are in the range of
200–700 mg/L, 50–500 mg/L and 10–12 mg/L, respectively (Acién
Fernández et al., 2018). The high COD levels also result in increased
turbidity, affecting light penetration in microalgal cultures. Ammonia
in its natural form is toxic to microalgae, affecting the oxygen evolving
complex of photosystem II and disruption of the electron gradient
across the thylakoid membranes (Gutierrez et al., 2016). Pharmaceu-
tical wastewater contains non-steroidal anti-inflammatory agents, an-
tibiotics, anti-depressants and anti-epileptics (Wang et al., 2016).
Heavy metals such as lead, copper, chromium, mercury and zinc are
most commonly seen in wastewaters. Heavy metals are known to in-
duce oxidative stress in microalgae by the production of free radicals,
inhibit photosynthesis by inactivation of essential enzymes and even
induce morphological changes (Posadas et al., 2017). Hence, the choice
of the microalgal strain depends on the characteristics of the waste-
water to be treated.
2.3. Microalgal cultivation reactors for wastewater treatment
Cultivation in open ponds are economically feasible and cost ef-
fective for large scale wastewater treatment. Raceway ponds with
mixing achieved by paddle wheels are the reactors of choice when it
comes to cost effective microalgal cultivation. This is particularly due to
the ease of installation and operation, low capital and maintenance
costs (10 €/m2), and low energy requirements (2–10 W/m3 wastewater)
(Acién Fernández et al., 2018). The use of municipal wastewater as a
nutrient medium in raceway ponds can reduce the production costs of
green algal biomass from 2.71 to 0.73 $/Kg biomass (Kang et al., 2015).
Wastewater based high rate algal ponds could be used for the produc-
tion of 40–70 tons of microalgal biomass per hectare per year, gen-
erating energy amounting to 800–1400 GJ energy per hectare per year
(Mehrabadi et al., 2015). Since the cost of microalgal cultivation is
offset by the wastewater treatment, the only energy and cost input re-
quired are for the biomass conversion process. Outdoor ponds utilize
sunlight as energy source, and long term exposure of the wastewater to
sunlight also help in disinfection of the treated wastewater. Sunlight
based ultraviolet light is essential for the disinfection and pathogen
removal of wastewaters in maturation ponds in addition to oxidative
damage caused by photosynthetic oxygenation (Craggs et al., 2014).
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Bioresource Technology 302 (2020) 122817
However, the biomass obtained in high rate algal ponds is almost al-
ways a consortium of microalgae and bacteria. Microalgae-bacteria
consortium is highly beneficial in organic carbon removal from high
COD wastewaters. Photosynthetic oxygenation provides continuous O2
supply for bacterial heterotrophic carbon metabolism, which in turn
produces continuous CO2 supply for microalgal photosynthesis. Such
symbiosis brings about effective carbon removal by bacteria with con-
comitant CO2 fixation and N/P removal by microalgae. Other beneficial
associations are a microalgal consortia with various nutrient and pol-
lution removal capabilities complementing each other and bringing
about highly effective wastewater treatment efficiency. The major issue
in open systems is the contamination of microalgal cultures by com-
petitive non-beneficial microalgae, parasites such as protozoans and
viruses and predators such as aquatic invertebrates (McBride et al.,
2014). Since it is not a closed system, the culture is also exposed to rain,
dust and other environmental debris. Contamination control in open
ponds is feasible by periodical management, but has not received much
research attention (Chisti, 2016).
2.4. Tolerance of microalgae towards economic outdoor cultivation
conditions
Another desirable quality in microalgae for wastewater treatment is
robust growth properties, in addition to the tolerance towards waste-
water conditions. As mentioned earlier, open ponds are the method of
choice for economic wastewater treatment, hence the chosen micro-
algal strain should be able to withstand the subtle to high changes in
temperature and illumination during day/night cycle and seasonal
variations. A temperature difference as high as 10° C can be observed
between day and night during spring and fall in countries like Taiwan.
Winter is most unsuitable for outdoor cultivation, and biomass pro-
ductivities along with nutrient removal are very low during winter
months (Godos et al., 2009). Enhanced pond systems treating municipal
wastewater showed higher biomass productivity and NH4-N removal
efficiencies in the high rate algal ponds in the summer months com-
pared to spring and winter (Sutherland et al., 2018; Sutherland et al.,
2014a). Also, high rate algal ponds promote the formation of large
microalgal-bacterial aggregates or flocs for settling and simple energy
efficient biomass harvesting. The enhanced pond systems in the above
mentioned study added two algal harvest ponds in continuous with the
high rate algal ponds for algal biomass recovery (Sutherland et al.,
2018).
2.5. Bio-prospecting/phyto-prospecting for isolation of robust native
microalgal strains
Screening of microalgae from native conditions (naturally occurring
wastewater algae in this context) is much more advantageous than
screening cultures from a laboratory collection or a culture center.
Phycoprospecting for native microalgal strains have the added benefit
of tolerance to the wastewater conditions resulting in effective nutrient
removal and growth properties suited to the native geological region
facilitating optimal outdoor cultivation (Wilkie et al., 2011). Chlorella
vulgaris, Neochloris oleoabundans and a native microalgal consortium
isolated from a wastewater treatment plant were applied for the
treatment of municipal wastewater. Higher nutrient removal efficiency
was achieved by the indigenous consortium with 63.2–80.8% inorganic
nitrogen removal, 30.8–70% phosphorus removal and 64.9–70.4% COD
removal (AlMomani & Örmeci, 2016). The dominant microalgal species
found in the wastewater were Tribonema, Scenedesmus, Microspora, Sti-
geoclomium, Oedogonium, Oscillatoria and Aphanocapsa. The distribution
of the microalgae varied with the wastewater treated (primary effluent,
secondary effluent, centrate) and the lag phase was considerably
shorter for the native algae compared to Chlorella vulgaris and Neochloris
oleaoabundans (AlMomani & Örmeci, 2016). Isolation of a 100 micro-
algal strains from local rivers and lakes were performed in Canada and
D. Nagarajan, et al.
they were screened in a high throughput method based on a micro titer
plate for nutrient removal and lipid rich biomass production in different
wastewaters (Abdelaziz et al., 2014). Six strains were able to grow at
10 °C in municipal wastewater and six other strains were able to grow
in municipal wastewater a 22 °C. Biomass productivities and lipid
content of the effective strains were in the range of 27.4–55.3 mg/L/d
and 17.2–45.0%, respectively (Mesdaghinia et al., 2015). Nine native
microalgal consortia were isolated from sewage treatment plant and
slaughterhouse effluent and were tested for their efficiency in treating
urban wastewater and high strength livestock wastewater (Choudhary
et al., 2016). The consortia PA6 was able to grow in both wastewaters
with nutrient removal efficiency in the range of 80–100% and a bio-
mass production of 1.93 g/L in livestock wastewater. The consortia was
rich in proteins (54%) with a theoretical methane potential of 0.79 m3
per kg volatile solids (Choudhary et al., 2016). Chlorella sorokiniana
pa.91 was isolated from dairy wastewater and could attain a specific
growth rate and biomass productivity of 0.375 day−1 and 0.233 g/L/d
in primary wastewater and 0.422 day−1 and 0.185 g/L//d in secondary
wastewater respectively. The nutrient removal efficiency for total ni-
trogen, ammonia, phosphate and COD were over 80% (Asadi et al.,
2019). Scenedesmus sp. RT_F isolated from a municipal wastewater
treatment plant was effective in treating secondary treated municipal
wastewater in a pilot scale reactor on-site the treatment plant. The NH4-
N, NO3-N and phosphate concentrations in the effluent were 0.05 mg/L,
0.40 mg/L, and 0.175 mg/L, respectively (Han et al., 2019). Chlorella
vulgaris, Chlorella saccharophila and Scenedesmus dimorphus were iso-
lated from eutrophic blooms and were effective in treating secondary
wastewater. As mentioned previously, the authors opined that it is not
necessary to acclimatize these strains for the local seasonal variations
(Camarena-Bernard & Rout, 2018). Chlamydomonas globosa, Chlorella
minutissima and Scenedesmus bijuga were isolated from carpet industry
wastewater and they were used as a consortium for the treatment of
untreated carpet industry rich wastewater (Chinnasamy et al., 2010).
The highest overall areal biomass productivity of 21.1 g/m2/d was
attained in 20–30 L polybags and a protein rich biomass (53.8%) was
obtained. The energy yield and the biomethane potential of the ob-
tained biomass were estimated to be 1265 GJ/ha/year and 12,128 m3/
ha/year, respectively with a total estimated energy recover via bio-
methane as 134,144 KWh/ha/year (Chinnasamy et al., 2010). Scene-
desmus sp. and Parachlorella sp. were isolated as co-flocculating mi-
croalgae from the rare earth mining effluent via continuous cultivation
for three years in the wastewater and subsequent transfer to nutritious
media (Zhang et al., 2019a). Even though laboratory cultures and ge-
netically engineered strains would be able to steer the microalgal
bioremediation in a highly beneficial way, the use of native strains
might decrease the release on invasive foreign microbes into the en-
vironment (Wilkie et al., 2011).
In summary, native strains acclimatized to the local seasonal var-
iations could perform better in wastewater treatment and current
technologies show that it is feasible to integrate microalgae based
treatment methods in the existing treatment methods to improve the
environmental benefits and procure beneficial biomass.
3. Nutrient and pollution removal efficiency of microalgae
The major goal of wastewater treatment is to remove the excess
nutrient content and pollutants of the wastewater prior to environ-
mental release. High nutrient content might lead to eutrophication of
the local fresh water and marine habitats, while release of pollutants
might lead to serious human and animal health effects due to possible
bioaccumulation via the food chain. In this section, the removal of
major nutrients and pollutants by microalgae is discussed in detail.
3.1. Carbon
Carbon constitutes for about 50–50% of the microalgal biomass and
5
Bioresource Technology 302 (2020) 122817
is the most essential macronutrient present in all the major cellular
components such as polysaccharides, amino acids, nucleic acids, and
lipids (Markou et al., 2014). Microalgae can fix atmospheric carbon via
photosynthesis in photoautotrophic mode. This requires an effective
light supply strategy and a gaseous or soluble CO2 supply. Treatment of
wastewaters by microalgae in photoautotrophic mode is hugely carbon
limited, as most of the organic carbon present in certain wastewaters
are not available for microalgal metabolism. This is the probable cause
for the relatively low COD removal efficiencies attained in microalgae
based wastewater treatment systems. Supply of atmospheric CO2
(which is about 0.04%) does not support optimal growth. Hence, a
supply of inexpensive gaseous carbon source is essential (Uggetti et al.,
2018). Organic carbon sources can also be assimilated by microalgae
via the mixotrophic or heterotrophic mode of growth. The carbohy-
drates/sugars produced by the dark reactions of photosynthesis in algae
are catabolized by a carbon metabolic pathway, providing energy and
carbon for growth and biomass accumulation. Wastewaters are rich in
organic carbon compounds such as acetate, propionate, ethanol, me-
thanol, butyrate, lactate and other volatile organic acids which could be
assimilated by microalgae (Lowrey et al., 2015). Chlorella sorokiniana
CCAP 211/8K could consume 0.74 g/L of acetate in three days under
dark conditions, and could outcompete bacterial growth in unsterilized
acetate rich dark fermentation effluent with a biomass production of
0.33 g/L (Turon et al., 2015). Other short chain fatty acids and alcohols
commonly seen in wastewaters and anaerobic digestion effluents can be
assimilated in varying degrees by microalgae and the utilization effi-
ciency is strain specific (Chen et al., 2018; Turon et al., 2016). How-
ever, microalgal growth and nutrient removal efficiencies are inhibited
by high organic loading and a dilution of the wastewater to reduce the
initial COD loading could reduce the inhibitory effects. Dilution of
anaerobically digested sterile piggery wastewater improved growth of
Chlorella vulgaris CY5, and the N-limited condition (achieved in 20
times dilution) resulted in an enhanced lipid accumulation of 54.7% by
dry weight (Marjakangas et al., 2015). Thus, microalgal growth could
be carbon limited in certain wastewaters, and mixotrophic/hetero-
trophic mode of cultivation is much suited for organic carbon removal
due to the alleviation of light supply issues in highly turbid wastewaters
(Lowrey et al., 2015).
3.2. Nitrogen
Nitrogen is an essential macronutrient for microalgal growth and
the inorganic/organic nitrogen present in wastewaters can be utilized
by microalgae. Nitrogen is essential for the synthesis of amino acids
which are the building blocks of proteins, and proteins are the quin-
tessential cellular machinery performing survival ensuring tasks such as
light harvesting, photosynthesis and energy generation in microalgae
(Grobbelaar, 2007). Microalgae are capable of assimilating up to 2–10
ton N/ha/year in low irradiation conditions and a high 5–25 ton N/ha/
year in high irradiance regions, when the photosynthetic efficiency is in
the range of 1–5% (Acién Fernández et al., 2018). The commonly used
inorganic nitrogen sources in microalgal cultivation in microalgae are
nitrate salts, nitrite salts and ammonia. Nitrate and nitrite salts are
eventually converted to ammonia before being assimilated into amino
acids via the glutamine synthetase/glutamate synthase pathway or the
glutarate dehydrogenase pathway. Thus, ammonium is the preferred
nitrogen source for microalgae, due to the reduced amount of energy
required for assimilation (Perez-Garcia et al., 2011). Microalgae can
attain 100% NH4-N removal in certain wastewaters, compared to total
nitrogen or nitrate/nitrite removal (Luo et al., 2016; Zheng et al.,
2019a). In the case of organic nitrogen sources, urea can be effectively
utilized by several microalgae. Spirulina platensis UTEX 1926 showed
better growth performance when urea was used as nitrogen source in
fed-batch cultures, compared to ammonium sulphate (Soletto et al.,
2005). Coccomyxa acidophila could use urea as both a carbon and ni-
trogen and could accumulate 3.55 g lutein per gram biomass in
D. Nagarajan, et al.
mixotrophic mode (Casal et al., 2011).
Amino acids can be readily utilized by certain microalgae as ni-
trogen source and reactions catalyzed by aminotransferases assimilate
the nitrogen from the amino acids and expels the carbon skeletons
(Hellebust and Ahmad, 1989). Glycine as a nitrogen source could
support the growth of over 80% of the algal strains tested (Berland
et al., 1979). Dunaliella viridis growth suppression by nitrogen depletion
could be rescued by only four amino acids (which could serve as a ni-
trogen source and hence restore growth): glutamine, histidine, cysteine,
and tryptophan. Of these, histidine was transported inside the cells,
while the other three amino acids were decomposed extracellularly in
the culture medium and the ammonium thus released was assimilated
(Murphree et al., 2017). Addition of 1 mM of glutamate nitrogen en-
hanced the biomass production and lipid accumulation of Scenedesmus
vacuolatus (Gupta et al., 2019). Thus, the hydrolytic product of proteins
– amino acids could be utilized by microalgae to some extent, aiding in
total nitrogen removal.
Other than these common nitrogen sources, wastewaters could
contain other complex nitrogen compounds that cannot be utilized or
metabolized by microalgae. In the conventional activated sludge sys-
tems and microalgae-bacteria consortium treating wastewaters, higher
total nitrogen removal rates are achieved by the bacterial ni-
trification–denitrification process which is a dissimilatory process
converting chemically bound nitrogen to nitrogen gas. This leads to a
loss of nitrogen to the atmosphere, and recovery of nitrogen is the vital
point in microalgae based processes. Insufficient light supply in deep
ponds using microalgae-bacterial consortia helps bacterial denitrifica-
tion outcompete microalgal nitrogen assimilation (Sutherland et al.,
2014b), hence sufficient light supply is key for effective microalgal
nitrogen assimilation. It was shown that higher nitrogen removal oc-
curred in mesocosm ponds (5 m2) that promote bacterial nitrification/
denitrification, compared to full scale ponds (1-ha) that promotes mi-
croalgae growth over bacteria via the photosynthetic advantage
(Sutherland et al., 2020). Addition of allylthiourea for inhibition of
bacterial nitrification in algal photobioreactors treating sewage water
resulted in an increase of NH4-N, which in turn supported higher mi-
croalgal biomass production (2.8 mg/L chlorophyll a) dominated by
Cryptomonas and Chlorella (Krustok et al., 2016). The control reactors
without allylthiourea showed an increased presence of NO3-N sug-
gesting bacterial nitrification and the dominant microalgae present
were Chlorella and Scenedesmus (1.6 mg/L chlorophyll a) (Krustok et al.,
2016). Other than these biotic methods of nitrogen removal, ammonia
volatilization at higher pH and oxygen levels typically achieved in high
rate algal ponds treating wastewater is another major method of ni-
trogen removal. Nitrogen removal by ammonia stripping accounted for
32–47% nitrogen removal followed by microalgal uptake and assim-
ilation and uptake at 25–26% in high rate algal ponds treating urban
wastewater (García et al., 2000) Thus, careful consideration of these
factors and minimizing nitrogen losses by abiotic or bacterial dissipa-
tion is essential for promoting higher microalgal growth and N recovery
in microalgae based wastewater treatment.
3.3. Phosphorus
Phosphorus is another essential macronutrient for microalgal me-
tabolism, due to its presence in the nucleotides, energy molecules, lipids
and polysaccharides. Phosphorus is generally supplied as phosphate
salts in the culture medium in excess of the nutritional requirements,
since phosphates can precipitate with the other metal ions in the
medium. Microalgae can assimilate most of the organic and inorganic
forms of phosphate after mineralization by phosphatases and conver-
sion to orthophosphates (Markou et al., 2014). Phosphate is the
common growth limiting factor for microalgal biomass production in
natural environments and altering P levels in water bodies are asso-
ciated with eutrophication or development of algal blooms (Patel et al.,
2012). Microalgae/cyanobacteria can uptake high levels of phosphate
6
Bioresource Technology 302 (2020) 122817
and accumulate as polyphosphate granules in insoluble from in a pro-
cess called luxury uptake. Luxury up take could account for about
53 ± 8% of the total cellular phosphate reserves (Schmidt et al.,
2016). High light intensity (150 μE/m2·s) promoted acid soluble
phosphate accumulation in synthetic wastewater containing
0.41–3.16% phosphates, treated by a Scenedesmus sp. dominated mi-
croalgal consortium (Powell et al., 2009). An algal biofilm isolated from
a rendering plant could remove P from wastewater at an initial con-
centration of 2.9 mg/L, with a removal efficiency of over 97% under
continuous illumination in a horizontal flat plate photobioreactor.
Under simulated natural light conditions (12 h:12 h light dark cycles),
the P removal efficiency decreased to 41% (Sukačová et al., 2015). The
microalgal population of the algal biofilm consisted of Phormidium au-
tumnale, Pseudanabaena sp., Scenedesmus acutus, Cymbella minuta and
Chroococcus sp.. The biomass productivity under continuous illumina-
tion was 12.2 g/m2/d, while natural light conditions reduced the bio-
mass productivity to 12.2 g/m2/d (Sukačová et al., 2015). The effect of
temperature on luxury uptake of phosphate is debatable; Powell et al
suggested that increased temperature increased phosphate uptake
(Powell et al., 2008), while Schmidt et al suggested that temperature
does not have a significant effect on phosphorus uptake (Schmidt et al.,
2016). High phosphate levels in the culture medium (5 mM) induced
luxury uptake of phosphorus, decreased starch synthesis and induced
lipid synthesis in Chlorella sp. (Zhu et al., 2015). The starch synthesis
decreased by 16.7% and the lipid synthesis decreased by 22.4% at
5 mM phosphate, compared to the normal phosphate levels of 0.17 mM.
Thus N-deplete and P-replete conditions could enhance lipid accumu-
lation in microalgae (Zhu et al., 2015). Nitrogen concentrations in
wastewater could influence P uptake and removal by microalgae. Both
Chlorella sp. and Scenedesmus sp. showed higher P removal when the N
levels in wastewater was higher. At 40 mg/L N concentration, P re-
moval was 6 mg P/L; but when the N concentration was reduced to
20 mg/L, P removal was reduced to 2 mg P/L for both Chlorella and
Scenedesmus (Beuckels et al., 2015). Thus an optimal N/P ratio is es-
sential for maximal P removal. Bacterial P removal in wastewater
treatment plants are mainly accomplished by Acinetobacter sp., Accu-
mulibacter and Tetrasphaera (Mielczarek et al., 2013). However, the
ecological relationship between microalgae and bacteria in P removal
needs to be addressed. Other than the biotic removal methods, abiotic P
removal can occur via precipitation at pH > 8.5 in the presence of
cations such as magnesium and calcium. Precipitation of calcium
phosphate and magnesium phosphate could account for up to 50% of
total P removal in microalgae based systems treating wastewaters
(Wang et al., 2017).
3.4. Sulfate
Sulfur is an essential macronutrient for microalgal cultivation and it
is required for the synthesis of sulfur containing amino acids, sulfated
polysaccharides and lipids. The sulfur content of microalgal biomass is
in the range of 0.15–1.6% and sulfate is the main sulfur source for
microalgal cultivation (Grobbelaar, 2007). Wastewaters containing
high amounts of sulfate, particularly mining industries, distilleries and
certain food processing industries are a potential health risk to humans
and animals. High sulfate content in drinking water (> 500 mg/L) can
cause mild laxative effects with dehydration and it imparts a bitter
medicinal taste to the water (Mohammadi et al., 2018). Sulfate in
concentration up to 271 mg/L promoted the growth of Chlorococcum sp.
GD, with enhanced N/P removal and self-flocculation. Deprivation of
sulfur resulted in poor nutrient removal capabilities and reduced floc-
culation (Lv et al., 2017). Sulfur deprivation can reduce photosynthetic
activity at high light intensities, generating temporary anoxic condi-
tions. This is permissible for the activation of the oxygen sensitive hy-
drogenases and results in hydrogen production in green algae (Oey
et al., 2016). Five microalgae species namely, Chlorella sp., Chlamydo-
monas sp., Oocystis sp., Scenedesmus sp., and Fischerella sp. were applied
D. Nagarajan, et al. Bioresource Technology 302 (2020) 122817

for the remediation of power plant wastewater with an initial sulfate
concentration of 5231 mg/L sulfate, 1858 mg/L sodium and a very low
COD of 165.5 mg/L. Under photoautotrophic conditions with ambient
air as a CO2 supply, Oocystis sp. attained the highest sulfate removal
efficiency of 32% with a biomass production of 530 mg/L, followed by
Chlamydomonas sp. with a sulfate removal efficiency and biomass
production of 26% and 420 mg/L, respectively (Mohammadi et al.,
2018). Dictyosphaerium sp. MM-IR2, isolated from a power plant was-
tewater attained a sulfate removal efficiency of 37.29% with an initial
sulfate concentration of 5637 mg/L and a COD of 165.5 mg/L
(Mohammadi et al., 2019).
3.5. Heavy metals
Heavy metals are present in mining industry, electroplating in-
dustry, tannery, dye and pigment industry, and pesticide industry
wastewaters. Heavy metals and their derivatives/metabolites are per-
sistent in the environment, and are toxic even at very low concentra-
tions. Direct exposure to heavy metals occur via exposure to heavy
metal containing wastewaters or via accumulation in the food chain.
Mercury toxicity index of marine fish is an appropriate illustration of
the disastrous effects of toxic metals bioaccumulation in the food chain.
The mode of action, toxic doses, metabolic profile and removal me-
chanism differs from each metal. Apart from the physical and chemical
methods, live biomass and powdered microalgae are applied for the
bioremediation of heavy metal containing wastewater (Table 1). Heavy
metal containing wastewater bioremediation with microalgae involves
one of the following process for detoxification: (i) bio adsorption on the
cell surface via electrostatic interaction as the cell surface has a
negative charge accomplished by complexation and micro precipita-
tion, (ii)biodegradation by enzymes released into the culture medium,
(iii) photo degradation promoted by the light exposure and photo-
synthetic oxygenation in open ponds for microalgal cultures, (iv) bio-
transformation into other metabolites that are non-toxic or easily re-
moved, and (v) bio sequestration or bioaccumulation into organelles
such as chloroplasts, vacuoles and mitochondria (Bulgariu and
Gavrilescu, 2015; Sutherland and Ralph, 2019). Once inside the cell,
the metal ions induce a stress response that will negate the toxic effects
of the metal ion like oxidative stress or chelate the metal ions with
metal binding proteins called metallothioneins (Priya et al., 2014). The
major heavy metals that pose a severe health risk include Hg (mercury),
As (arsenic), Pb (lead), Cr (chromium), Cd (cadmium), Zn (zinc), Ni
(nickel), Cu (copper), Mo (molybdenum) and B (boron) (Bulgariu and
Gavrilescu, 2015). The removal efficiency of these metals by microalgae
is summarized in Table 1. This demonstrates the potential of microalgae
for removal of heavy metal pollutants from wastewater. Since bio-
sorption seems to be the major removal mechanism, the proper disposal
of the algal biomass obtained after treatment is crucial to prevent
secondary pollution from mismanaged sludge.
Tannery wastewater from the leather processing industries is a
major source of heavy metal containing wastewater. An indigenous
Scenedesmus sp. AUBAC-002 isolate was used to treat tannery waste-
water under photo-autotrophic conditions. The removal efficiency for
Cr, Cu, Pb and Zn were 81.2–96%, 73.2–98%, 75–98% and 65–98%,
respectively. A biomass of 11.8x106 cells/ml, with nitrate and phos-
phate removal efficiency of 44.3% and 95% was observed (Ajayan
et al., 2015). Scenedesmus sp. was used for the treatment of wet market
wastewater and a TOC, TN, TP, Fe and Zn removal efficiency of

Table 1
Heavy metal removal efficiency of various microalgae species.
Heavy metal Microalgae Removal capacity Reference

Cr(VI), Cu(II), Pb(II) and Cd(II) Fe2O3/Synecdatehocystis sp. PCC6803 composite Cr(VI) (69.77 mg/g) Pb(II) (62.63 mg/g) Cd(II) (Shen et al., 2020)
(42.12 mg/g) Cu(II) (38.68 mg/g)
Zn, Mn Desmodesmus sp. MAS1 Heterochlorella sp. MAS3 40–80% Fe (Abinandan et al.,
40–60% Mn 2019)
Cu, Mo in mine tailings water Chlorella vulgaris 64.7% Cu (Urrutia et al., 2019)
99.9% Mo
B, Mn and Zn in flue gas Consortium composed of Desmodesmos sp., Chlorella sp., 46.8 ± 9.45 g/L B, 253.66 ± 40.62 g/L Mn (Aslam et al., 2019)
Scenedesmus sp. and filamentous cyanobacteria 355.5 ± 50.69 g/L Zn
Hg(II), Cd(II) and Pb(II) Alginate immobilized Chlamydomonas reinhardtii 0.53 mmol/g Hg(II) (Bayramoğlu et al.,
0.71 mmol/g Cd(II) 2006)
1.49 mmol/g Pb(II)
Hg Chlorella sp. DT expressing Bacillus megaterium mercuric Reduction of Hg2+ to elemental Hg0 (Huang et al., 2006)
reductase gene
Hg containing synthetic dental Chlorella vulgaris biomass powder 85.88% (Fard & Mehrnia, 2017)
wastewater
Hg(II) from contaminated Transgenic Chlamydomonas reinhardtii, ultrasound 28% (He et al., 2011)
sediments assisted
Arsenite, arsenate Scenedesmus sp. Bioaccumulation of 606–761 μg/g dry weight (Bahar et al., 2013)
biomass
Arsenic (III, V) C. minutissima As(III) – 290 mg (Arora et al., 2017)
As(V) – 330 mg
Arsenic (III, V) Scenedesmus sp. IITRIND2 As(III) – 360 mg (Arora et al., 2017)
As(V) – 370 mg
Pb(II) Spirogyra sp. 140 mg metal/g of biomass (Gupta & Rastogi, 2008)
Cd, Pb, Ni, Zn Lyngbya taylorii – unmodified dry biomass 1.47 mmol/g Pb (Klimmek et al., 2001)
0.37 mmol/g Cd
0.65 mmol/g Ni
0.49 mmol/g Zn
Cd, Pb, Ni, Zn Lyngbya taylorii – phosphorylated dry biomass 2.52 mmol/g Pb (Klimmek et al., 2001)
3.08 mmol/g Cd
2.79 mmol/g Ni
2.60 mmol/g Zn
Pb Spirulina maxima – intact biomass powder 84% (Gong et al., 2005)
Pb Spirulina maxima – calcium treated biomass powder 92% (Gong et al., 2005)
Pb(II) Free Chlorella sorokiniana 108.04 mg/g biomass (Akhtar et al., 2004)
Pb(II) Sponge immobilized Chlorella sorokiniana 123.67 mg/g biomass (Akhtar et al., 2004)

Footnote: B – boron, Cr – chromium, Cu – copper, Cd – cadmium, Hg – mercury, Mn – Manganese, Mo – molybdenum, Ni – nickel, Zn – zinc.

7
 Table 2
Summary of microalgae based bioremediation of different wastewaters: nutrient removal and biomass production.
Wastewater
Primary clarified municipal
wastewater
Anaerobically digested
municipal wastewater
Untreated municipal
wastewater
Synthetic tertiary municipal
wastewater
Anaerobically-digested black
water
Anaerobic digestion effluent
from cattle manure
Synthetic acid mine drainage
wastewater with 1–4 g/L
sulfate
8
Power plant wastewater
Power plant wastewater
A mixture of 19% treated
effluent and 21% digestate
Pulp and paper mill effluent
Anaerobically digested pulp
and paper mill effluent
Slaughterhouse wastewater
Cattle slaughterhouse
wastewater
Microalgal strain Treatment conditions Biomass production Nutrient removal Remarks Reference
Galdieria sulphuraria Outdoor 700L raceway reactors aerated with 2% – In effluent: P < 1 mg/L Biomass energy recovered effectively by (Li et al., 2019)
D. Nagarajan, et al.
CO2, influent wastewater conditions: NH4-N: BOD5:30 mg/L hydrothermal liquefaction
22.7–29.2 mg/L PO43−:2.1–3.9 mg/L, NH4-N:19.5–19.9 mg/L
BOD5:111.5 mg/L
C. vulgaris FACHB-8 1L glass photobioreactor, 25 ± 2 °C, light 1.62 g/L, 99.21 mg/L/d COD: 83% Highest lipid content of 26.4% achieved (Xu et al., 2019)
intensity of 3000–3500 lx, 12-h light/12-h dark productivity TN:96.5% at alternating high-low temperatures
cycle, CO2 supply at 15 L/h NH3-N:97.8%
TP: 99.2%
Algal consortium, PA6: Outdoor 100L pilot scale attached film 3.48 ± 0.44 g/m 2/d COD: 53 ± 2% Lipid content of 35.2%, hydrothermal (Naaz et al., 2019)
Phormidium and Chlorella bioreactor, HRT 6 days productivity NH3-N: 81 ± 3% liquefaction was found to be energy
pyrenoidosa NO3-N: 81 ± 3% effective conversion
TP: 75 ± 2%
Chlorella sorokiniana Indoor photobioreactors, 130–150 rpm agitation, 700 mg/L COD:17–47% BOD: 60–80%TN: 42% increase in carbohydrates and 13% (Rani et al., 2019)
25 °C, continuous illumination at 90 μmol/m2/s, 12–100%NO3- increase in proteins
HRT 1–6 days depending on inoculum size N:53–96%TP:59–92%
Chlorella sorokiniana and Outdoor 211 L tubular PBR inside a green house, – TN: 28–62 mg/L/d High bacterial nitrification resulted in (Silva et al., 2019)
Chlorococcum sp. 25 °C, light intensity at 60–270 μmol/m2/s TP:2.3–5.4 mg/L/d ≥1000 mg NO2-N/L, biomass suitable
for fertilizer application
Scenedesmus sp. L-1 250-mL Erlenmeyer flasks, 10% effluent, white 4.65 g/L COD: > 90% Lipid productivity of 81.90 mg/L/day, (Luo et al., 2019)
fluorescent light at 5000 lx, light/dark cycles of NH3-N: > 90% 60.52% monounsaturated fatty acids,
12 h/12 h, 25 °C, batch culture NO3-N: > 90% 39.48% saturated fatty acids
TP: 79–88%
Microalgae-bacteria Revolving algal biofilm reactor, continuous – COD: > 90% Sulfate removal rate of 0.56 g/L/d and a (Zhou et al., 2018)
consortium operation, 3 days HRT, light illumination at NH3-N: 52–69% removal capacity of 6.47 g/m2/d for the
130 μ mol/cm2/s SO4: 35–46% algal bioreactor
TP: 79–88%
Oocystis sp. Erlenmeyer flasks (50 ml), 25 ± 1 °C, 530 mg/L 25.24 mg/L/d SO4: 32% – (Mohammadi et al.,
fluorescent lamp illumination at 4150-lx 2018)
intensity, 16:8h light–dark cycle, 30 days.
Chlamydomonas sp. Erlenmeyer flasks (50 ml), 25 ± 1 °C, 420 mg/L20 mg/L/d SO4: 26% – (Mohammadi et al.,
fluorescent lamp illumination at 4150-lx 2018)
intensity, 16:8h light–dark cycle, 30 days.
Algal-bacterial 1L glass PBR, batch, 8 days, 20 °C, light intensity 21.5 g/L/d productivity TOC: 63.7% – (Moreno-Garcia
consortium dominated by of 80 μmol/m2/s, light/dark photoperiod of NH3-N: 71% et al., 2019)
Chlorella sp. 12 h/12 h, 130 rpm agitation. NO3-N: 63.23%
PO4-P:94.6%
Mixed algal culture with Outdoor 30L open circular ponds, batch-30 days, – COD:75% Biomass composition consisted of: (Usha et al., 2016)
two Scenedesmus sp. 23 ± 3 °C and 250–800 μmol/m2/s, 60% BOD:82% protein – 16 ± 2.45% carbohydrate –
wastewater NO3-N: 65% 18.90 ± 3.2% lipids – 15.8 ± 4%, iron
PO4-P: 71.29% – 2.48 mg/g, zinc – 0.366 mg/g
Scenedesmus acuminatus 1-L glass bottle PBRs, 5% CO2 supply at a flow 8.22 g/L volatile COD: 36% Biomass rich in carbohydrates: 6.05% by (Tao et al., 2017)
SAG 38.81 rate of 0.105 L/min, light intensity at 240 μmol/ suspended solids NH3-N: 99% dry weight.
m2/s, 22 ± 2 °C, 11–12 days PO4-P:96.9%
Mixed algal culture Lab scale 0.8L PBR, batch – 7 days, agitation 2048 g/L TOC: 89.6%TN:70.2%TP: 96.2% – (Taskan, 2016)
220 rpm, 23 ± 1 °C, ambient sir supply at
300 ml/min, light intensity at 100 μE/m2/s, 12/
12 h light/dark conditions
Phormidium sp. Bioreactor, batch mode, 128 h. dark conditions, 0.43 mg algal sludge per COD: 90% Biomass suitable for biodiesel production (Maroneze et al.,
20 °C, pH 7.6, aeration 1 vvm, mg COD TN:57% 2014)
TP: 52%
(continued on next page)
Bioresource Technology 302 (2020) 122817
 Table 2 (continued)
Wastewater
Aquaculture wastewater
Shrimp culture wastewater
Palm oil mill effluent
Acid and heat treated palm oil
mill effluent
Tertiary petrochemical
wastewater
Molasses wastewater with
48–56% total sugars
Raw and anaerobically
digested food processing
industry wastewater
9
Dairy wastewater
Tannery wastewater
Mixture of piggery and winery
wastewater
Anaerobically digested starch
processing wastewater
Textile industry wastewater
Textile industry wastewater
Microalgal strain Treatment conditions Biomass production Nutrient removal Remarks Reference
Chlorella sp. GD 1 L in column-type glass-PBRs, 26 ± 1 °C, 1.296 g/L/d COD: 80% Effective CO2 fixation from boiler flue (Kuo et al., 2016)
D. Nagarajan, et al.
300 μmol/m2/s, 2% CO2 supplied at 0.2 vvm, TN:90% gas at 2.333 g/L/d
batch – 7 days NH3-N: 77%
NO3-N: 83%
TP: 99%
Chlorella sorokiniana MB- 1-liter glass PBR with added BG-11 medium, 3.5 g/L, 1.3 g/L/d – Biomass with a lutein content and lutein (Chen et al., 2019)
1 M12 26 °C, 300 rpm agitation, 300 μmol/m2/s, productivity productivity of 3.89 mg/g and 5.0 mg/L/
mixotrophic mode with 6 g/L sodium acetate, d
75% wastewater, 0.5% salinity, semi-continuous
operation
Chlamydomonas sp. UKM Erlenmeyer flasks 2L, 25 °C, light intensity at 0.634–0.917 g/L COD: 8.59–29.13% – (Ding et al., 2016)
6. 20,000 lx, light/dark cycle of 16:8 h, aeration TN: 43.5–72.97%NH3-N:
with 2% CO2 at 0.7 vvm, batch – 9 days 58.58–100%
TP: 38.15–63.53%
Chlorella sorokiniana CY-1 1 L glass PBR, 25 °C, 300 rpm agitation, light 2.12 g/L COD: 47.09% Biomass with a lipid content of 11.21% (Cheah et al., 2018)
intensity at 8000 lx, 2.5% CO2 aeration at TN: 62.07%
0.1vvm, batch-15 days TP: 30.77
Tribonema sp. Vertical tubular PBRs, 25 °C, 300 μmol/m2/s, 5.9 g/L COD: 97.8% Biomass with 37.5% lipids and 45.6% (Huo et al., 2018)
1.5% CO2 aeration at 0.5 vvm, TN: 100% carbohydrates
TP:100%
Scenedesmus sp. Z-4. 250 ml Erlenmeyer flasks, 25 °C, light intensity at 3.5 g/L COD: 87.2% Biomass had a lipid content of 28.9% (Ma et al., 2017)
3000 lx with a light/dark cycles of 12 h/12 h, TN: 90.5% and a lipid productivity of 94.4 mg/L/d
TP: 88.6% was attained
Scenedesmus obliquus 500 ml Erlenmeyer flasks, 29 °C, light intensity 211 mg/L/d COD: 89% A lipid productivity of 27.5 mg/L/d was (Gupta and Pawar,
NCIM 5586) 80 μmol/m2/s at with a light/dark cycles of 10 h/ productivity TN: 84% obtained. 2018)
14 h, ambient air at supply at 0.1 vvm, CO2 TP: 70%
purging thrice a day for pH maintenance at 7–8.
Scenedesmus quadricauda 1L glass PBRs, 25 °C, light intensity 90 μmol/m2/ 0.36–0.43 g/L TOC:76.77% – (Daneshvar et al.,
s at with a light/dark cycles of 12 h/12 h, TN:92.15% 2019)
ambient air supply, batch – 12 days, two stage TP:100%
mixotrophic cultivation. SO4:100%
Scenedesmus sp. 1L flask, 25 °C, light intensity 182.5 μmol/m2/s 0.9 g/L COD: 80.33% – (da Fontoura et al.,
at with a light/dark cycles of 12 h/12 h, ambient NH4-N:85.63% 2017)
sir supply at 1 L/min, batch-24 days, 88.4% TP: 96.78%
wastewater
Chlorella sp. MM3 250 ml flasks, 23 °C, continuous illumination at 5.1x106 cells/ml TN:51–89% Lipid accumulation in biomass reached (Ganeshkumar
200 μmol/m2/s, batch-10 days, piggery:winery TP:26–49% 29–51%. et al., 2018)
wastewater ratio 20:80
Chlorella pyrenoidosa, Outdoor airlift circulation photobioreactor with a 0.37 g/L/d COD: 65.99% Temperature fluctuation influenced the (Tan et al., 2014)
FACHB-9 dynamic membrane filtration, continuous CO2 TN: 83.06% saturation level of fatty acids in lipid,
supply at 5–9% from functional anaerobic TP: 96.97% maximum lipid content achieved was
digester 19.95%
Scenedesmus abundans 1L flask PBR, illumination at 440 W with a light 4.79 g/L BOD: 13.9% – (Brar et al., 2019)
NCIM No. 2897 dark cycle of 16:8h, 25 °C, batch – 25 days, 75% COD: 86.8%
textile wastewater NO3-N:68.8%
PO4-P: 70.79%
Cl-:44.4%
Chlorella pyrenoidosa 1L flask PBR, illumination at 440 W with a light 3.97 g/L BOD: 24.05% – (Brar et al., 2019)
NCIM 2738) dark cycle of 16:8h, 25 °C, batch – 25 days, 75% COD: 85%
textile wastewater NO3-N:74.43%
PO4-P: 28.01%Cl-:61%
(continued on next page)
Bioresource Technology 302 (2020) 122817
D. Nagarajan, et al. Bioresource Technology 302 (2020) 122817

Footnotes: BOD – biological oxygen demand, COD-chemical oxygen demand, TOC-total organic carbon, TN-total nitrogen, NO3-N-nitrate nitrogen, NH4-N-ammonia nitrogen, TP-total phosphorus, PO4-P-phosphate
80.34%, 65.32%, 76.77%, 65.76% and 82.12%, respectively (bte Jais
et al., 2015). A heterotrophic Botryocossuss sp. NJD-1 was applied for
effective Cr(VI) removal from synthetic organic containing wastewaters
(Zheng et al.,

(Shen et al., 2019). The chromium concentration was 5 mg/L and the
Reference

2019b)

organic carbon sources methanol, ethanol and sodium acetate were

used in different concentrations. In the presence of 5 mg/L Cr(VI), the
TOC, TN, TP and Cr(VI) removal efficiency were 98.2%, 66.9%, 99.2%
and 94.2% respectively. The removal mechanism was shown to be the
pH controlled cultures can alleviate

reduction of Cr(VI) to Cr(III) and complexation on the cell surface as

identified by image analysis (Shen et al., 2019). Another interesting
outcome of this microalgal reduction of heavy metals is the possible
formation of metal nanoparticles. Selenastrum capricornutum and Mi-
crocystis aeruginosa with cadmium adsorbed on the cell surface, when
ammonia toxicity.

exposed to selenium sources, CdSe nanoparticles were formed (Zhang

et al., 2019b). The nanoparticles possessed a definite spherical structure
with fluorescence under UV illumination, which was quenched by ex-
Remarks

posure to Hg2+. Thus, the biofabricated CdSe nanoparticles could be

used as an “on–off” sensor for mercury, with a detection limit at
0.021 µM mercury (Zhang et al., 2019b). In summary, microalgae are
capable of heavy metal removal in various wastewaters, mainly via bio
adsorption and biotransformation and the critical issue is the disposal of
metal loaded microalgal biomass.
The potential of microalgae for the removal of organic micro pol-
Nutrient removal

lutants such as antibiotics and steroidal hormones were discussed in

NH4-N:100%

detail in the authors’ earlier review on swine wastewater bioremedia-

COD: 99%

TP: 95%

tion by microalgae (Nagarajan et al., 2019). The information provided

in this section vouches for the nutrient and pollutant removal efficiency
of microalgae. Microalgae based bioremediation of different waste-
waters are summarized in Table 2. It can be seen that a vast spectrum of
wastewaters could be treated by microalgae; this is by no means an
Biomass production

exhaustive list, but it provides an overall view of the diversity of the

wastewaters that could be treated by microalgae, alongside beneficial
products production such as lipids, carbohydrates, proteins and pig-
3.83 g/L

ments.

4. Potential bottlenecks in microalgae based wastewater

200 μmol/m2/s, batch-7 days, C/N ratio of 25:1,
5L closed PBR, 28 °C, continuous illumination at

treatment and future research perspectives

As discussed in the previous sections, microalgae based wastewater

treatment is effective for both bioremediation of wastewater and re-
covery of energy/nutrients from wastewater. The major factors af-
fecting microalgae based wastewater treatment and possible research
perspectives are discussed in detail.
Treatment conditions

Wastewater treatment efficiency is greatly influenced by the in-

herent characteristics of the wastewater to be treated and the micro-
algal species used (Posadas et al., 2017). Influent wastewater char-
acteristics such as COD, BOD, TN, TP, other heavy metals and trace
elements, pH, solids content, turbidity, micro-pollutants content differ
pH 7

phosphorus, SO4-sulfate, Cl- -chloride, PBR-photobioteactor.

for each wastewater and is highly inconsistent. It is challenging to si-

mulate a synthetic wastewater for laboratory experimental purposes
Chlorella vulgaris FACHB-

due to the complex nature of wastewaters, since they contain additional

unidentified components that might affect the outcome of the process
Microalgal strain

(Delrue et al., 2016). In addition, cultivation conditions such as tem-

perature, pH, carbon supply and illumination will influence biomass
productivity, and in turn will affect nutrient removal efficiencies. The
number of variables to be considered for optimization of microalgal
30

biomass production and effective nutrient removal to meet the dis-

charge standards are several. Design and development of computer
aided simulation programs are needed for effective design of an optimal
process for attaining maximal biomass productivity.
Manure free piggery
Table 2 (continued)

The choice of the seed inoculum for wastewater treatment is crucial

in treatment efficiency. As it can be seen in Table 2, many microalgae
wastewater

and cyanobacteria are capable of effective nutrient removal and re-

Wastewater

mediation of wastewaters with beneficial products production. How-

ever, the presence of bacteria in the culture will result in a symbiotic
relationship between them as discussed previously in Section 2, and

10
D. Nagarajan, et al.
enhance the treatment efficiency. Microalgae consortia with different
individual capabilities can complement each other and bring out the
best treatment efficiency. High throughput screening methods can
speed up the bio prospecting aspect (Abdelaziz et al., 2014; Delrue
et al., 2016), while acclimation of an isolated microalgae/consortia to
the selected wastewater conditions can also be applied. Multi-omics
analysis of the microalgal metabolism under stress conditions will help
in the development of potential strains for beneficial nutrient recovery
from wastewater (Wan et al., 2019). Most of the genetic engineering
studies are focused towards microalgae based biofuel production, and
research focus on microalgal physiological changes during wastewater
treatment is essential. Genome analysis of the three Chlorella strains
Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp.
Dachan revealed that the best performing strains for piggery waste-
water treatment - BD08 and Dacahan possess enriched nitrogen and
phosphorus assimilation capabilities as well as certain gene alteration
such as chromosome inversion (Wu et al., 2019). The symbiotic bac-
terium associated with Dachan strain, Microbacterium chocolatum
played a synergistic role in enhancing the nutrient removal rates and
biomass productivity of Dachan strain (Wu et al., 2019). Genetic ana-
lysis of the high performing strains could provide valuable insights into
the strain dependent efficiency of wastewater treatment.
A great majority of the wastewater treatment studies using various
microalgae are performed in laboratory conditions. Other than certain
studies reporting high rate algal ponds and microalgae-bacteria con-
sortium (de Godos et al., 2010; Matamoros et al., 2015), pilot scale
studies with axenic microalgal cultures are few. A pilot scale plant for
treating primary treated municipal wastewater in open paddle wheel
driven ponds was simulated in Fukushima, Japan. A total of 10 ponds
occupying one hectare of land was applied as the treatment reactor, and
preliminary laboratory experiments using a consortium of indigenous
Scenedesmus sp. and Desmodesmus sp. were performed (Sasongko et al.,
2018). Simulations using primary treated municipal wastewater and
flue gas as carbon source indicated that for initial TN and TP con-
centration of 42.7 mg/L and 5.8 mg/L, a net carbon sequestration of
82.77–140.58 tons per hectare per year could be attained, with con-
comitant production of 63–107 tons per hectare per year. Hydrothermal
liquefaction with the wet microalgal paste was identified as the best
option for preparing bio-crude oil based on energy requirements. En-
hancing biomass productivity and technological advancement in
downstream processing were identified as the potential challenged for
economic feasibility of the pilot scale plant operation (Sasongko et al.,
2018). A 5-ha high rate algal pond demonstrated effective primary
settled wastewater treatment in New Zealand with BOD5, NH4-N, re-
active P and pathogenic E. coli removal efficiencies of 50%, 65%, 19%
and 2 log, respectively (Craggs et al., 2012). In case of the use of
photobioreactors, a semi-open photobioreactor with a surface area if
11.9 m2 was constructed at the Metro Plant, USA for treating centrate
with a light supply of 25 μmol/m2/s. CO2 injection helped reduce
bacterial blooms and the Chlorella sp. grew predominantly in mixo-
trophic mode. A biomass production of 34.6 g/m−2/day TSS and
17.7 ± 4.2 g/m−2/day VSS was attained with removal efficiencies of
70%, 61% and 61% for COD, total Kjeldahl nitrogen and soluble P,
respectively (Min et al., 2011). Techno economic evaluation of a pilot
scale treatment plant for dairy effluent showed that a treatment plant
with > 1 million liters per day treatment capacity for a period of
20 years is feasible with an internal rate of return of 118% in a payback
period of 1.9 years. Algal biomass harvesting by centrifugation was
determined as the most cost incurring part and an annual biomass
production of 504 ton with a production cost of $0.482/Kg and
240,000 m3 of treated clean effluent (Kumar et al., 2020). Thus, large
scale microalgae based wastewater treatment is feasible and much re-
search attention is needed on pilot scale studies, life cycle analysis and
techno economic analysis.
Effective illumination of the microalgal culture is essential for op-
timal photosynthetic activity, maximal nutrient removal efficiency and
11
Bioresource Technology 302 (2020) 122817
corresponding biomass accumulation. Light intensity, spectral quality
and the source of light energy are the determinants of the photo-
synthetic efficiency of algal cells (Le et al., 2019). In photobioreactors,
self-shading of cells, absorbance of excess light by surface cells and
dissipation as heat energy, existence of dark zones that perform re-
spiration over photosynthesis affects total biomass production. How-
ever, exposure to high light intensity leads to photo oxidative damage
of the photosynthetic apparatus resulting in photo-inhibition (Carvalho
et al., 2011). In raceway ponds (which is the reactor of choice), the
hydrodynamics of the culture fluid induced by the paddle wheel mixing
results in constant movement of the cells, and with the given turbidity
of the culture, exposure of the cells to light for effective photosynthesis
is a complex phenomenon (Demory et al., 2018). Also, the depth of the
raceway reactors determines the light exposure, since deep ponds have
dark zones that are not exposed to light promoting bacterial activity.
For effective nutrient removal by microalgae, pond depth should be
maintained at < 0.2 m (Acién Fernández et al., 2018). A microalgal
consortium consisting of Chlorella sp., Scenedesmus sp., and Stigeoclo-
nium sp., was used for the treatment of municipal wastewater in a high
rate algal pond of varying depths (0.2, 0.3 and 0.4 m) (Kim et al.,
2018). The pond with a depth of 0.2 m showed the best performance of
all with a biomass productivity, nitrogen removal, phosphorus removal
and settleability of 6.16 g/m2/d, 82.5%, 89.7% and 96.4% respectively.
The biomass productivity was 38% higher compared to 0.3 and 0.4 m
ponds (Kim et al., 2018). Thus, pond depth is an important factor
governing light exposure, nutrient removal and biomass accumulation
in wastewater high rate algal ponds.
The major operational difficulties of the existing microalgae based
treatment systems for wastewater treatment is the high hydraulic re-
tention time applied, as long as 7–10 days and the land requirement
which is about 10 m2 per capita. Optimal and cost effective treatment
methods must operate at an HRT of < 1 day with a per capita land
requirement of 1 m2 (Acién Fernández et al., 2018). The longer HRT is a
prerequisite for effective decontamination of potential micro pollutants,
but an HRT of 4 days has been reported to be sufficient for the removal
of endocrine disruptors in a high rate algal pond (Matamoros et al.,
2015). Land availability is projected as a major challenge for integra-
tion of microalgae based systems with the current wastewater treatment
plants (Roostaei and Zhang, 2017). A cost effective and area efficient
high volume V-shaped pond was designed for pilot scale studies for
treating dairy wastewater in India. The inverted pyramid shape pro-
vided an increased surface area of 4 m2 for better light capture with a
3 m2 culture volume (Kumar et al., 2020). Such innovative designs are
needed for cost effective wastewater treatment and alleviate land use
change effects. Sutherland et al suggested that effective nitrogen re-
moval and optimal microalgal biomass productivity occurred in a pilot
scale pond size ponds (330 m2) compared to full scale (1 ha) ponds
(Sutherland et al., 2020). Thus, integration of pilot scale ponds to
currently existing treatment facilities might also prove fruitful. Also, the
location of the treatment plants is vital. It is unmanageable to install
and operate a wastewater treatment plant in urban areas, while trans-
port of the wastewater to remote locations is expensive. A balance is
required, since the plant location must have good exposure to sunlight
and far from populated areas due to odor related issues. Industrial
wastewaters could be treated by in-house treatment plants, which could
be beneficial. An in-farm anaerobic digester is a common fixture in
most livestock farms to treat the wastewater generated.
In the currently used urban wastewater treatment methods, COD/
BOD removal is commendable, but N and P removal is not effective.
This is the reason why microalgae based methods are recommended for
secondary and tertiary urban wastewater treatment. However, it is
worth noting that high organic matter loading is inhibitory towards
microalgal growth (Wang et al., 2016), even though the N and P re-
moval efficiencies are far better than the activated sludge system. The
turbidity caused by high organic carbon content inhibits light pene-
tration, affecting photosynthetic efficiency. Also, most carbon present is
D. Nagarajan, et al.
in insoluble form or biologically unavailable for microalgal metabolism.
Algal growth also releases certain external organic matter that increases
the COD levels of wastewater (Wang et al., 2016). Chlorella sp. and
Micractinium sp. grown in N-rich, P-limited wastewater (primary mu-
nicipal wastewater and sludge centrate) leads to the production of high
amounts of protein rich extracellular polysaccharides, as high as
60 mg/L proteins and 40 mg/L carbohydrates after sonication assisted
extraction (Wang et al., 2014). For effective COD removal using mi-
croalgae, either dilution of the wastewater to lower COD loading or a
pretreatment for organic carbon removal would be beneficial.
Nutrient removal in open microalgal ponds could also occur via
volatilization of ammonia and phosphate precipitation induced by the
alkaline pH and oxygenation occurring due to photosynthesis (Posadas
et al., 2017). The allocation of nitrogen and phosphorus between as-
similation by microalgal biomass, biological dissipation by bacterial
activity and abiotic removal by stripping determines the effective re-
covery of nutrients in the algal biomass. Many studies investigated the
symbiotic relationship between microalgae and bacteria for nitrogen
removal, but such studies related to phosphorus removal are rare. This
could be due to the reason that phosphorus levels are usually low and
most of the times > 90% removal could be achieved. Nutrient loss by
abiotic/biotic dissipation needs to be minimized and maximal recovery
by the microalgal biomass must be obtained.
Algal biomass harvesting is the most cost incurring part in any
microalgae based technology. This is mainly due to the small size of the
microalgal cells, a comparable density as that of water, negative
charges on their surface areas, and low density cell cultures obtained in
wastewater treatment processes under outdoor conditions (Singh and
Patidar, 2018). Harvesting and drying/dewatering accounts for up to
30% of the microalgae production costs (Acién Fernández et al., 2018).
Cost effective harvesting techniques like flocculation is preferable
(Vandamme et al., 2013), and the choice of self-flocculating strains
could alleviate the excess chemical addition to achieve flocculation
(Spilling et al., 2011).
Depending on the cultivation mode and wastewater composition, a
supply of inorganic carbon might be needed. Carbon is essential for
microalgal growth and inorganic carbon can be supplied as either
gaseous CO2 or carbonate salts. The benefit of sparging gaseous CO2 is
multifold: the decrease in pH as the result of CO2 addition inhibits non-
biological N and P removal such as volatilization and precipitation and
the sparging of the gas provides agitation which provides mixing of the
culture and prevents settling of the cells. Sparging of a high rate algal
pond treating urban wastewater showed enhanced biomass growth of a
Scenedesmus sp. dominated microalgal consortium by 66–100%, prob-
ably by alleviating the negative effects of the wastewater and ammonia
toxicity (Uggetti et al., 2018). Industrial flue gases (de Godos et al.,
2010; Ji et al., 2015) and fermentation off-gases (Laude et al., 2011;
Zhang et al., 2017) are rich in CO2 and are an inexpensive source of
gaseous CO2. Thus, wastewater bioremediation and carbon sequestra-
tion can be achieved in a single step.
The added value potential of the microalgal biomass obtained in a
wastewater treatment process is crucial in determining the economic
feasibility of the process. Hydrothermal conversion processes are sui-
table for biomass (either a microalgal consortium or a microalgae/
bacterial consortium) that are not rich in carbohydrates/lipids
(Goswami et al., 2019). Protein rich biomass can be applied as animal/
aquaculture feed additives due to their nutrient content (Delrue et al.,
2016). Thermochemical conversion technologies can yield gaseous
biofuels and biochar. Whole microalgal biomass and microalgal bio-
mass can be used as adsorbents for certain pollutants and heavy metals.
Thus, complete biomass valorization is crucial for cost effective was-
tewater treatment.
Possible environmental impacts of the microalgae based wastewater
treatment system in case of GHG emissions, energy input Vs energy
output, net energy gain, nutrient recovery efficiency, water footprint
and fossil fuel depletion by energy consumption need to be analyzed.
12
Bioresource Technology 302 (2020) 122817
Life cycle analysis indicated that environmental impacts of the con-
ventional activated sludge based treatment system was two to five times
higher that of natural systems such as high rate algal ponds (Garfí et al.,
2017). In addition, high rate algal ponds in warm climatic conditions
were shown to be economically feasible for the treatment of wastewater
from small communities compared to activated sludge system, and in-
tegration of bio fertilizer production might improve the energy re-
covery of the obtained microalgal biomass (Arashiro et al., 2018).
5. Conclusions
Wastewaters are an abundant secondary source for finite resources
like phosphorus and therefore nutrient removal from wastewaters is
crucial in a circular bioeconomy standpoint. Microalgae-based nutrient
removal from wastewaters serves the dual purpose of bioremediation of
wastewaters and beneficial algal biomass production. Open field re-
actors under outdoor conditions are cost and energy effective for was-
tewater treatment. A careful balance of cultivation conditions is es-
sential for preventing nutrient loss by abiotic removal. Techno
economical and life cycle analysis of microalgal treatment methods and
integration of pilot scale demonstration plants with the existing treat-
ment facilities will empower resource efficiency driven economic
growth.
CRediT authorship contribution statement
Dillirani Nagarajan: Investigation, Writing - original draft. Duu-
Jong Lee: Writing - review & editing. Chun-Yen Chen: Resources,
Validation. Jo-Shu Chang: Supervision, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge financial support received from
Taiwan’s Ministry of Science and Technology under grant number
MOST 108-3116-F-006-007-CC1, 108-2218-E-029-002-MY3, 107-2221-
E-006-112-MY3, 108-2621-M-006-020, and 108-2218-E-006-00.
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