ALGAL-00734; No of Pages 13

Algal Research xxx (2016) xxxxxx

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Algal Research

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A review on the use of microalgal consortia for wastewater treatment

Ana L. Gonalves, Jos C.M. Pires, Manuel Simes
LEPABE, Departamento de Engenharia Qumica, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

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

Article history: The development of anthropogenic activities has led to an excessive disposal of wastes into water bodies, thus
Received 31 March 2016 reducing water quality and damaging aquatic ecosystems. To avoid the negative impacts associated to the dis-
Received in revised form 16 October 2016 charge of wastes into water courses, effective remediation processes are required to reduce nitrogen and phos-
Accepted 8 November 2016
phorus concentrations in discharged efuents. Current methodologies applied for nutrients removal tend to be
Available online xxxx
complex, expensive and energy demanding. Therefore, cultivation of microalgae has appeared as an emerging al-
Keywords:
ternative for nutrients removal from wastewaters. These photosynthetic microorganisms require large amounts
Microalgal-bacterial consortia of nitrogen and phosphorus for their growth. However, since it is very difcult to maintain pure cultures of these
Microalgal consortia microorganisms in wastewater treatment processes, several studies have reported the use of natural and articial
Microbial interactions microalgal consortia composed exclusively by microalgae or by microalgae and bacteria. The use of these consor-
Nutrients removal tia in the remediation of wastewaters can be very advantageous because: (i) cooperative interactions between
Wastewater treatment the co-cultivated microorganisms can occur, enhancing the overall uptake of nutrients; and (ii) these systems
tend to be more resistant to environmental conditions oscillations. This study provides an updated review of
the literature regarding the application of microalgal consortia in the remediation of wastewaters from different
sources, focusing on the mechanisms involved in nutrients removal by microalgae and the main interactions
established between the microorganisms integrating the consortia and how they can inuence nutrients removal
efciencies.
2016 Elsevier B.V. All rights reserved.

1. Introduction followed by nitrication and denitrication [3,7]. However, several an-

In the last decades, large amounts of wastewaters have been pro-
duced, mainly due to anthropogenic activities, such as agricultural prac-
tices, urbanization and industrialization [14]. The continuous disposal
of wastewaters without adequate treatment can pose serious pollution
problems. One of the major problems associated to the continuous dis-
charge of efuents into water bodies is the so called eutrophication phe-
nomenon the enrichment of water resources in nutrients, mainly
nitrogen and phosphorus. This phenomenon is responsible for the de-
velopment of algal blooms, spread of aquatic plants, oxygen depletion
and loss of key species, resulting in the complete degradation of fresh-
water ecosystems [3,5]. Besides the degradation of freshwater ecosys-
tems, development of these blooms can be considered a public health
risk for the surrounding populations [6]. This clearly evidences the
need for effective treatment methods, able to reduce nitrogen and phos-
phorus concentrations in wastewaters before discharging into natural
bodies.
Nitrogen and phosphorus removal in wastewaters are mainly re-
moved in the tertiary treatment phase (Fig. 1). The most commonly
used methods include biological processes, such as anaerobic digestion
Corresponding author.
E-mail address: mvs@fe.up.pt (M. Simes).
aerobic and nitrication and denitrication cycles are required to
achieve the nutrient levels accepted by EU legislation. Additionally,
these methods require several tanks and internal recycles of activated
sludge, resulting in an overall increase of process costs, complexity
and energy input [811]. Alternatively, nitrogen and phosphorus re-
moval may be achieved by chemical methods, such as precipitation
using aluminium and iron salts. However, these methods are costly
and produce large amounts of sludge contaminated with chemical com-
pounds [1214], requiring further treatment.
To overcome the drawbacks associated to the commonly used tertia-
ry treatment methods, biological treatment using microalgae (a general
term commonly applied to refer to photosynthetic microorganisms,
such as eukaryotic microalgae and prokaryotic cyanobacteria) has
been extensively studied in the last decades. Microalgae require large
amounts of nitrogen and phosphorus for their growth [3,15], meaning
that these microorganisms can effectively uptake nitrogen and phos-
phorus from wastewaters. In fact, high nitrogen and phosphorus remov-
al efciencies (80100%) from wastewaters of different sources (e.g.
agricultural, industrial and municipal) have already been reported for
microalgae [1620]. Additionally, the use of microalgae for nutrients re-
moval presents several advantages [13]: (i) nitrogen and phosphorus
assimilated by microalgae can be recycled by the production of fertil-
izers from microalgal biomass; (ii) the resulting biomass can be used

http://dx.doi.org/10.1016/j.algal.2016.11.008
2211-9264/ 2016 Elsevier B.V. All rights reserved.

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
2 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx

Fig. 1. Main steps involved in wastewater treatment processes and currently applied methods [2,4].

for the production of bioenergy, food, animal feed and pharmaceuticals;
and (iii) an oxygenated efuent is discharged into the water bodies.
More recently, several studies have reported the potential of
microalgal consortia (microalgal and microalgal-bacterial) in different
applications, including biomass production and nutrients removal [2,
15,2124]. The use of polycultures for nutrients removal purposes can
be very advantageous since combining microorganisms with different
metabolic activities and adapted to different environmental conditions
was found to allow the development of a robust biological system that
can operate under different environmental conditions and nutrient
loads [2527]. Additionally, cooperative interactions can be established
between the microorganisms integrating the consortia, which can result
in higher nutrient uptake rates [3]. Accordingly, this manuscript high-
lights the advantages of using microalgal consortia in the bioremedia-
tion of wastewaters, providing an updated overview of successful
consortia already applied in this eld.
limitations in terms of overheating, difculties in scale-up and higher
construction costs [37]. The most commonly used PBRs include at
plate reactors, bubble-column reactors and tubular reactors [36,38,39].
Microalgal production in open systems is less expensive in terms of con-
struction and operation and has a larger production capacity [37,40,41].
However, due to insufcient mixing, oscillations in the culture condi-
tions and higher susceptibility to contaminations, biomass productiv-
ities and nutrients removal efciencies are lower than those achieved
in closed PBRs. Additionally, these systems are more prone to CO2 diffu-
sion to the atmosphere, evaporative losses of water and poor light utili-
zation by cells [37,38,41,42]. Open systems can be divided into two
categories: natural ponds, which include lakes, lagoons and ponds,
and articial ponds or containers [36,38,42]. The most commonly used
systems include shallow big ponds, tanks, circular ponds and raceway
ponds [38,42,43]. The growth of microalgal consortia in suspended cul-
tivation systems has already been reported in the literature. Cultivation

2. Microalgal culturing Table 1

Advantages and limitations of suspended- and immobilized-cells cultivation systems in
Microalgal cultivation for wastewater treatment purposes can be wastewater treatment processes.
performed in suspended- or immobilized-cell systems [20,2830].
Cultivation
Table 1 presents the main advantages and limitations of these systems systems Advantages Limitations
in wastewater treatment. For an effective cultivation and remediation
Suspended-cells Widely studied and opti- Microalgal harvesting prior
process, some parameters, such as light, temperature, pH, nutrients sup- cultivation mized to the disposal of the treat-
ply and mixing should be carefully controlled [3134]. systems Larger amounts of waste- ed wastewater is required
water can be processed
2.1. Suspended-cells cultivation systems Applicable in large scale
operations
Immobilized-cells Microalgal harvesting prior High costs associated to the
Cultivation in suspension is the most commonly used form cultivation to the disposal of the treated polymeric matrix (in the
for microalgal growth [35]. Systems typically used for microalgal systems wastewater is simpler case of cell entrapment)
growth in suspension include closed or open bioreactors. Closed Aging cultures are more High surface area required
protected against (in the case of microalgal
photobioreactors (PBRs) for microalgal growth can be more advanta-
photoinhibition adhesion and biolm for-
geous because (i) culture conditions and growth parameters, such as Immobilization matrix con- mation)
pH, temperature, mixing, carbon dioxide (CO2) and oxygen (O2) con- fers cells higher resistance Light limitation may occur
centrations, can be strictly controlled; (ii) evaporation and contamina- to harsh environments, such Applicable only for small
tions can be easily avoided; and (iii) higher cell concentrations can be as salinity, metal toxicity and pilot scale operations
and pH
achieved [3638]. Despite these advantages, PBRs have some

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx
of a microalgal-bacterial consortium consisting of the cyanobacterium
Nostoc palusodum Kutz and the bacterium Rhizobium galegae in a
suspended cultivation system has demonstrated to be stable for
18 months and to maintain its structure and activity in seed treatment
of Galega orientalis [44].
2.2. Immobilized-cells cultivation systems
Although suspended-cells cultivation systems have been effectively
applied in wastewater treatment, further separation of microalgal bio-
mass is required, as it may contribute to 6090% of the efuent biolog-
ical oxygen demand (BOD) [45]. To overcome the problems associated
to the time-consuming and energy-demanding harvesting methods
currently applied for microalgal separation, cultivation systems based
on microalgal immobilization have appeared as an alternative to the
suspended-cells cultivation systems [35,4547]. According to Tampion
and Tampion [48], an immobilized cell consists in a cell that by natural
or articial techniques is prevented from moving independently of its
neighbours to all parts of the aqueous system in study. Natural or pas-
sive immobilization occurs through the innate ability of microalgal
cells to attach to a specic surface, resulting in biolm formation. On
the other hand, articial or active immobilization techniques include
adsorption, connement in liquid-liquid emulsions, capturing with
semi-permeable membranes, covalent coupling and entrapment within
polymers [46,4951]. The most commonly used methods for microalgal
immobilization include cell entrapment within a polymeric matrix, nor-
mally alginate and carrageenan, and cell adhesion and biolm forma-
tion in a solid surface [46,5052]. In cell entrapment, microalgal cells
are conned in the polymeric matrix and substrates and products dif-
fuse to and from the cells through the pores present in the matrix. The
use of these systems already demonstrated enhancement in biomass
and pigment productivities and lipid contents [46,51,52]. However,
the high costs associated to the immobilization matrix can be a limiting
factor when the aim is to process large amounts of wastewater. Regard-
ing algal biolms, it is thought that if enough surface area is provided,
microalgal growth can be higher than in suspended-cells cultivation
systems. These systems result in biomass productivities similar to
those reported for cell entrapment at reduced costs: algal biolm forma-
tion presents lower water requirements and does not require an expen-
sive immobilization matrix [52]. Currently, microalgal immobilized
systems are used for different applications, such as metabolites produc-
tion, hydrogen and electricity production, toxicity measurements and
remediation processes [30]. Paniagua-Michel and Garcia [53] have
immobilized natural microbial mats obtained from marine sediments
on glass wool to promote nitrogen removal from shrimp culture efu-
ents. The immobilized mat consisted of lamentous forms of
cyanobacteria (Microcoleus chthonoplastes, Spirulina sp., Oscillatoria sp.,
Schizothrix sp., Calothrix sp. and Phormidium sp.), green algae (Chlorella
sp. and Dunaliella sp.), diatoms (Nitzchia sp. and Navicula sp.) and nitri-
fying bacteria (Nitrosomonas sp. and Nitrobacter sp.) which was able to
effectively remove nitrate-nitrogen (NO3-N) and ammonium-nitrogen
(NH4-N). After 20 days of culture, NO3-N and NH4-N removal efcien-
cies were 95 and 97%, respectively. The immobilization of a
microalgal-bacterial consortium constituted by species of Chlorella,
Scenedesmus, Stichococcus and Phormidium and by the bacteria
Rhodococcus sp. and Kibdelosporangium aridum onto ceramics, capron
and wool has resulted in the formation of a stable consortium,
preventing the involved microorganisms from being washed off [54].
Additionally, this system promoted an effective removal of phenols,
heavy metals (copper, nickel, zinc, manganese and iron) and chemical
oxygen demand (COD). In the study performed by Chavan and Mukherji
[55], a stable consortium was obtained when growing species of
Phormidium, Oscillatoria and Chroococcus and the oil-degrading bacteri-
um, Burkholderia cepacia on a rotating biological contactor. This consor-
tium has also demonstrated to be effective in petroleum hydrocarbon
removal.
Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
3
2.3. Factors inuencing microalgal growth
Microalgal growth can be inuenced by several factors, both biotic
and abiotic. Biotic factors include the presence of pathogens, such as
bacteria, fungi and viruses, and the competition by other microalgae,
whereas abiotic factors include light (quality and quantity), tempera-
ture, pH, salinity, nutrient qualitative and quantitative proles, dis-
solved oxygen concentration and the presence of toxic compounds.
Additionally, microalgal growth can be inuenced by operational condi-
tions, such as hydraulic residence time, harvesting rates, gas transfer
and mixing, since these parameters control CO2 availability, shear
rates and light exposure [3134].
Photoautotrophic growth of microalgae is driven by light supply, as
this is the energy source that is used to convert inorganic carbon, usually
CO2, into organic carbon. For light irradiances below the light saturation
point, photosynthetic activity is proportional to light irradiance. Howev-
er, for high light irradiance values, which may differ according to the
microalgal species used and to other culturing parameters, particularly
temperature, photosynthetic receptor system can be damaged,
resulting in the inhibition of photosynthesis and therefore, microalgal
growth [41,56,57].
Another parameter that strongly inuences microalgal growth is
temperature [58]. Optimal temperature for microalgal growth is species
specic [59]: (i) some species, commonly found in polar environments,
tolerate temperatures below 10 C [60,61]; (ii) others grow in moderate
temperatures (1020 C); and (iii) others, from tropical regions, tolerate
temperatures above 30 C [62]. However, an increase in temperature
normally results in an increased metabolic activity, whereas lower tem-
peratures contribute to the inhibition of microalgal growth [59,63]. Ide-
ally, microalgal cultures should be maintained at temperatures similar
to those observed in the environments where they were collected [10,
32].
Regarding the effect of pH, the majority of microalgal species are
grown in a pH range between 7.0 and 9.0 [3234]. However, some
microalgae are alkalophilic, whereas others are acidophilic. For exam-
ple, the microalga Spirulina platensis is able to grow in environments
with a pH ranging between 9.0 and 10.0 [64], while the microalga
Chlorococcum littorale prefers acidic environments, with pH values
ranging between 5.0 and 6.0 [65,66]. The pH of the culture medium is
responsible for physiological changes in microalgae and, therefore, it is
crucial to maintain microalgal cultures in the optimal range to avoid cul-
ture loss by extreme pH values. Additionally, culture medium pH can be
associated to the supplied CO2 concentration, due to the chemical equi-
libria established between the following species: CO2, H2CO3, HCO3 and
CO23 [3234]. The increase of CO2 concentration in gaseous input
stream results in a decrease in pH values in the culture. Therefore, the
CO2 fed to microalgal cultures should be strictly controlled, so that
microalgal cells are not affected by a decrease in the pH of the culture
medium. On the other hand, during the cultivation time, it is common
to observe an increase in pH, due to CO2 uptake [33,34]. Thus, daily
monitoring of the pH in the culture medium is very important to assess
microalgal growth and to dene procedures to maintain the cultures in
the optimal pH range.
In the case of salinity, optimal levels differ according to microalgal
species [32,34]. Modications in the salinity of the culture medium
can have adverse effects on microalgal growth and composition, due
to (i) osmotic stress; (ii) ion (salt) stress; and (iii) alterations in the
membrane permeability to ions [67]. Evaporative losses and rainfalls
(in open systems) are the main contributors to modications in culture
medium salinity. The negative effect of these alterations on microalgal
growth has been extensively discussed by Tredici and Materassi [68]
and Garca-Gonzlez et al. [69]. The control of salinity levels can be per-
formed by adding either freshwater or salts to the culture medium [70].
Regarding nutrients supply, the main nutrient required for autotro-
phic microalgal growth is inorganic carbon, since it is the precursor of
photosynthetic reactions. However, microalgal growth is dependent
4 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx

on other nutrients, such as nitrogen and phosphorus: these nutrients
are required for the synthesis of nucleic acids and proteins. Their pres-
ence at limiting concentrations can result in reduced growth rates and
biomass productivities. The most commonly forms of inorganic nitrogen
supplied are NO3-N, NH4-N and urea [7174]. Concerning phosphorus
uptake, this nutrient is required in the form of soluble phosphates and
should be supplied in large quantities, since not all the phosphorus com-
pounds are bioavailable for microalgae [31,33]. Apart from these nutri-
ents, commonly referred as macronutrients, microalgal growth still
requires the presence of trace elements, particularly metals, such as
Mg, Ca, Mn, Zn, Cu and Mo [33,71]. Additionally, some studies have re-
ported the use of vitamins to improve microalgal growth [33].
Finally, mixing is a key growth parameter in microalgal culture be-
cause it allows an equal distribution of light and nutrients among
microalgal cells, thus avoiding the existence of stagnant zones, and im-
proves gas transfer between the culture medium and the air [31,33,75,
76]. Gas transfer should not be compromised, since the air bubbled
into the cultures contains the CO2 required for photosynthesis and
removes the produced oxygen. Mixing of microalgal cultures is also im-
portant in preventing microalgal settling [31,33,75] and in avoiding
thermal stratication [31]. Selection of an appropriate method depends
on the scale and used cultivation system.
3. Wastewaters as nutrients source
As referred in Section 2.3, the main nutrients required for microalgal
growth are carbon, nitrogen and phosphorus. Since wastewaters pres-
ent considerable concentrations of these nutrients, several studies
have reported the potential of microalgal growth in wastewaters from
different sources: (i) domestic [77,78]; (ii) leachate [79,80]; (iii) agricul-
tural [16,81,82]; (iv) renery [83]; and (v) industrial [54,84]. These
studies have revealed that using wastewaters for microalgal cultivation
promotes an effective treatment of these waters (nitrogen and phos-
phorus removal efciencies reported correspond to 6099 and 54
95%, respectively); while contributing to the production of microalgal
biomass at reduced costs and with lower environmental impacts,
since nutrients supply and freshwater are not required [8587]. Addi-
tionally, microalgal cultivation in wastewaters attracts a worldwide at-
tention, because in a long term the use of chemical fertilizers as
nutrients source will become unsustainable, particularly in the produc-
tion of low-cost products, such as biofuels. However, up to now, only
about 30% of published works refer the use of wastewaters as culture
medium for microalgae, whereas the remaining 70% refer the use of
chemical fertilizers as nutrients source.
3.1. Characterization of different wastewaters
Wastewaters can be dened as disposable liquids or water-carried
waste resulting from domestic, agricultural, urbanization and industrial
practices. Usually, wastewaters may contain large quantities of oxygen
demanding wastes, pathogenic organisms, organic pollutants, nutrients,
such as nitrogen and phosphorus, inorganic compounds and sediments
[4]. Table 2 presents typical compositions of wastewaters from different
sources in terms of nitrogen, phosphorus and organic carbon, demon-
strating that wastewater composition strongly depends on its source.
In general, wastewaters from the swine industry present higher nitro-
gen and phosphorus levels than municipal wastewaters. On the other
hand, wastewaters resulting from brewery, starch and dairy industries
and potato-processing wastewaters present high concentrations of sol-
uble COD. To consider the use of microalgae in wastewater treatment,
the composition of the efuent to be treated should be previously
assessed, as nitrogen to phosphorus molar ratios (N:P) strongly inu-
ence microalgal biomass production and hence, nutrients uptake. Ac-
cording to the average elemental composition of microalgal biomass,
N:P molar ratios lower than 5:1 result in nitrogen limitation, whereas
N:P molar ratios higher than 30:1 result in phosphorus limitation [10].
N:P molar ratios determined for the wastewaters described in Table 2
range between 1:1 (for brewery and starch industry wastewaters) and
42:1 (for landll leachate). With N:P molar ratios of 9:1, 11:1, 14:1
and 15:1, piggery industry and domestic wastewaters, as well as dairy
manure and municipal sewage anaerobically-digested wastewaters
are considered the most appropriate for microalgal growth. In fact,
microalgal nutrients removal from domestic and piggery industry
wastewaters has been extensively reported in the literature [6,77,81,
8890].
3.2. Mechanisms involved in nutrients removal by microalgae
To improve wastewater remediation processes using microalgae, it
is very important to understand the mechanisms involved in nutrients
removal. Table 3 summarizes the mechanisms involved in carbon, nitro-
gen and phosphorus removal by microalgae.

Table 2
Typical compositions of different wastewaters already used for microalgal growth in terms of nitrogen, phosphorus and carbon.

Wastewater type Source Nitrogen (mg L1) Phosphorus (mg L1) Carbon (mg L1) Ref.

Agricultural wastewater Potato-processing efuent 54 NO3-N 48 PO4-P 745 COD [81]

12 NH4-N
Rice efuent 2595 TN 1294 PO4-P 25786480 COD [7]
Anaerobically-digested wastewater Dairy manure b1 NO3-N 240 TP 48554945 COD [104]
12791961 NH4-N
Municipal sewage 646 NH4-N 101 PO4-P 76 TOC [77]
Piggery manure 303495 NH4-N n.s. n.s. [90]
Industrial wastewater Brewery 211 NO3-N 57326 TP 5657837 COD [134]
3106 NH4-N
Carpet mill 028 NO3-N 2035 PO4-P 1412 COD [113]
1826 NH4-N
Dairy b1 NO3-N 35350 TP 200020213 COD [104,107,135,136]
120350 NH4-N
Oil, metal and chemical 1.9 NO3-N n.s. 1200 COD [54]
1.1 NH4-N
Piggery 324656 NH4-N 117 PO4-P 1247 TOC [89,90]
Starch 49115 NH4-N 50385 TP 247015440 COD [137]
Wood-based pulp and paper n.s. n.s. 1248 COD [84]
Municipal wastewater Domestic sewage 2566 NH4-N 712 PO4-P 400500 COD [11,77,126]
Landll leachate 112192 NH4-N 79 PO4-P 37254861 COD [80]
Sewage 1 NO3-N 112 PO4-P 183380 COD [81,87,102,103,130,131,136,138140]
23219 NH4-N

NO3-N - nitrate-nitrogen (mg N L1); NH4-N - ammonium-nitrogen (mg N L1); TN - total nitrogen (mg N L-1); PO4-P - phosphate-phosphorus (mg P L1); TP - total phosphorus
(mg P L1); TOC - total organic carbon (mg C L1); COD - chemical oxygen demand (mg O2 L1); n.s. - not specied.

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx 5

Table 3 NH4-N, in a two-step process catalysed by the enzymes nitrate reduc-

Mechanisms involved in nutrients (carbon, nitrogen and phosphorus) removal by tase and nitrite reductase [31,98,99]. In the rst step (Eq. 2), nitrate
microalgae.
reductase catalyses the reduction of NO3-N into NO2-N using nicotin-
Nutrients Mechanisms Cell incorporation amide adenine dinucleotide phosphate (NADPH) as reducing agent.
Carbon Further reduction into NH4-N is catalysed by nitrite reductase, which
CO2 Integration in the Calvin cycle Diffusion (5.0 b pH b 7.0) or uses ferredoxin to catalyse the six-electron transfer reaction (Eq. 3)
active transport (pH N 7.0) [31,99]:
Organic carbon Integration in the respiration Diffusion or active transport
metabolism (depending on molecules
Nitrate reductase
size) NO
3 2H 2e

NO
2 H2 O 2
Nitrogen
Nitrite reductase
N2-N Fixation by prokaryotic NO
2 8H 6e

NH
4 2H2 O 3
microalgae (cyanobacteria) into
ammonia, followed by
conversion into amino acids NH4-N resulting from NO3-N and NO2-N reduction and actively in-
NO3-N and Reduction into ammonium, Active transport corporated into microalgal cells is directly converted into amino acids
NO2-N followed by conversion into
via the glutamine synthetase-glutamate synthase pathway, where glu-
amino acids
NH4-N Direct conversion into amino Active transport tamine synthase catalyses glutamine formation from glutamate and
acids adenosine triphosphate (ATP), according to Eq. 4 [31,98,99]:
Stripping due to volatilisation n.a.
(high pH values and Glutamine synthase
temperatures) Glutamate NH
4 ATP Glutamine ADP Pi 4

Phosphorus
PO4-P Phosphorylation Active transport
Chemical precipitation (high pH n.a.
values and dissolved oxygen
concentrations)
n.a. - not applicable.
3.2.1. Carbon
Through photosynthesis, microalgae can x CO2 from both the at-
mosphere or ue gas emissions. Additionally, microalgae are able to up-
take soluble carbonates as a source of CO2. For low pH values (ranging
between 5.0 and 7.0), CO2 uptake occurs through diffusion. On the
other hand, for pH values higher than 7.0, bicarbonate (HCO 3 ) is the
most common form of inorganic carbon present in solution, which en-
ables the external carbonic anhydrase and promotes active transport
of this carbon source into microalgal cells [9193]. Once inside the
cells, HCO3 is converted into CO2 that can be xed by rubisco (ribulose
biphosphate carboxylase oxygenase), producing two molecules of 3-
phosphoglycerate [92,93]. Although microalgae are mainly autotrophic,
some microalgae are heterotrophic, using only organic carbon (e.g. ace-
tate, glucose, glycerol and ethanol) as carbon source, whereas others are
mixotrophic, using facultatively an organic carbon source in addition to
CO2 [9496]. In this growth regime, both respiratory and photosynthetic
mechanisms can occur [97].
3.2.2. Nitrogen
The most common inorganic nitrogen forms include nitrate, nitrite,
nitric acid, ammonium, ammonia, molecular nitrogen, nitrous oxide, ni-
tric oxide and nitrogen dioxide [31]. Microalgae play an important role
in both nitrogen xation and assimilation. Prokaryotic microalgae
(cyanobacteria) are able to x atmospheric molecular nitrogen (N2-
N), converting it into ammonia-nitrogen (NH3-N), which can either be
incorporated into amino acids and proteins or excreted to the environ-
ment [6,31]:
Nitrogenase
N2 8H 8e 16 ATP 2NH3 H2 16 ADP 16Pi
Eukaryotic microalgae, in turn, are able to assimilate xed nitrogen,
such as NH4-N, NO3-N and nitrite-nitrogen (NO2-N). These nitrogen
sources enter microalgal cells through active transport at the plasma
membrane. NO3-N, which is the most oxidized form, is thermodynami-
cally more stable than NH4-N and, hence, it is more common to nd this
inorganic nitrogen form in aquatic environments [31,71]. However, as-
similation of this nitrogen source requires previous reduction into
Since NH4-N assimilation does not require previous reduction steps,
it is thought that this is the preferred nitrogen form for microalgae.
However, according to Grobbelaar [71], the effect of both NO3-N and
NH4-N on microalgal growth is not well dened, since no signicant dif-
ferences were observed in terms of microalgal productivity. In addition
to microalgal uptake, NH4-N removal may occur in response to an in-
crease of pH and temperature, where large amounts of NH4-N can be
volatilized [1,6,100].
3.2.3. Phosphorus
Energy transfer and nucleic acid synthesis are mediated by phospho-
rus. This nutrient enters microalgal cells through active transport at the
plasma membrane in the forms of H2PO 2
4 and HPO4 . Incorporation of
PO4-P into organic compounds is performed through the following pro-
cesses: (i) phosphorylation at the substrate level; (ii) oxidative phos-
phorylation; and (iii) photophosphorylation. In these processes ATP is
produced from adenosine diphosphate (ADP) and an energy input,
which can be obtained from the oxidation of the respiratory substrates
or from the electron transport system of the mitochondria (in the case
of the rst two processes) and from light energy transformation (in
the case of the third step). The general reaction is represented by Eq. 5
[101]:
Energy
ADP Pi ATP 5
PO4-P removal can also be ruled by environmental conditions, such
as pH and dissolved oxygen concentration. For pH values above 8.0
and high oxygen concentrations, phosphorus precipitation may occur
[1,6,100,102].
3.3. Factors inuencing nutrients removal by microalgae
The pH of microalgal cultures constitutes one of the most important
factors inuencing nutrients removal. For example, the carbon concen-
trating mechanisms adopted by microalgal cells strongly depend on the
1
pH, as this parameter determines CO2 solubility in the culture medium.
Additionally, high pH values are responsible for NH4-N stripping and
PO4-P precipitation. However, other factors, such as light and tempera-
ture, also play an important role in nutrients uptake by microalgae. In
general, an increase in light supply results in increased nutrients remov-
al. Regarding the temperature effect, higher metabolic activities and
hence nutrients uptake rates can be observed for increased tempera-
tures, reaching its maximum when cultivation temperature is close to
the optimal values reported for the microorganisms in study.

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
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6 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx
Additionally, an increase in temperature lowers the solubility of some
nutrients, such as CO2 and NH4-N.
4. The role of microalgal consortia in nutrients removal
Although microalgae have been successfully applied in nutrients re-
moval from different wastewaters, it is difcult to maintain a microalgal
monoculture in these processes. Accordingly, several studies have re-
ported the advantages of using microalgal consortia over single-species
cultures (Table 4) [15,23,90,103,104]. Complex degradation processes,
which would be difcult to accomplish using monocultures, can benet
from the use of microalgal consortia. Additionally, application of these
consortia may result in the development of a robust system, able to re-
sist to environmental uctuations and invasion by other species [23,
105]. These consortia can naturally occur in the environment or be arti-
cially engineered, by combination of microorganisms that do not nec-
essarily co-occur, for a specic purpose [106]. Among the different
possibilities of consortia that can be established, the most commonly
used for wastewater treatment include microalgal consortia, which
are constituted exclusively by photosynthetic microorganisms (eukary-
otic and/or prokaryotic), and microalgal-bacterial consortia, which are
constituted by photosynthetic microorganisms and heterotrophic bac-
teria. The next sections describe the type of interactions that can be
established between the microorganisms integrating these consortia
and how these interactions can improve nutrients removal. Table 5
summarizes the main characteristics of both consortia.
4.1. Microalgal consortia
Interactions between photosynthetic microorganisms are not well
documented in the literature [107]. It is thought that growing these mi-
croorganisms in a consortium may result in both cooperative and com-
petitive interactions. On the one hand, these microorganisms may
establish cooperative interactions through the exchange of metabolites,
resulting in an overall increase in biomass productivities and hence, nu-
trients removal efciencies [108]. On the other hand, co-cultivation of
photosynthetic microorganisms may result in the excretion of second-
ary metabolites, also known as allelochemicals, that exhibit a negative
effect on the co-cultivated microorganisms [108110]. For example,
when growing a microalgal consortium composed by C. vulgaris and
Pseudokirchneriella. subcapitata, Fergola et al. [111] have demonstrated
that P. subcapitata growth was strongly inhibited by chlorellin, a fatty
acids mixture excreted by the co-cultivated C. vulgaris. Allelochemicals
production may be enhanced or suppressed by both abiotic and biotic
factors. The most important abiotic factors that enhance allelochemicals
production include nutrients starvation, low light intensities and tem-
peratures and high pH values. On the other hand, excessive nutrients
Table 4
Advantages and limitations of using microalgal consortia in wastewater treatment
processes.
Advantages Limitations
Robust systems (able to resist to envi- Wide variety of possible microalgal
ronmental uctuations and invasion combinations
by other species) Difculties in the consortia design
Broad specicity to different nutri- (microbial selection, ratio between
ents (the combination of microor- microorganisms, etc.)
ganisms with different nutrients Difculties in maintaining the con-
requirements results in the removal sortia in longer processes and in
of multiple nutrients at the same open systems
time)
Cooperative interactions result in in-
creased removal efciencies
Can be used in replacement of sec-
ondary treatment phase (the
microalgal-bacterial consortia), re-
ducing the oxygenation costs and
CO2 emissions
Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
Table 5
Main characteristics of microalgal and microalgal-bacterial consortia.
Microalgal consortia Microalgal-bacterial consortia
Established interactions deciently Well studied interactions
documented Cooperative and competitive interac-
Cooperative and competitive inter- tions can occur
actions can occur Nutrients exchange is the basis for
Can be applied in the tertiary treat- cooperative interactions
ment step of wastewater treatment Can be applied in both secondary and
Promote an effective removal of tertiary treatment steps of wastewa-
nitrogen, phosphorus and other ter treatment
elements, such as heavy metals Promote an effective removal of
nitrogen, phosphorus and organic
carbon, reducing the oxygenation
costs associated to the secondary
treatment step
concentrations, high light intensities and temperatures and low pH
values can inhibit allelochemicals production. Biotic factors inuencing
allelochemicals production are the concentrations of the involved mi-
croorganisms (the ones producing toxic compounds and target cells)
[108]. In wastewater treatment processes, interactions between photo-
synthetic microorganisms can have the following advantages: (i) en-
hancement of the overall nutrients uptake, providing that sufcient
nutrients are supplied; (ii) resistance to contaminants and predators
through the induction of allelochemicals production; and (iii) the devel-
opment of a settleable system (by combining single cell microorganisms
with occulating ones), thus avoiding the requirements for a harvesting
method. Additionally, the use of microalgal consortia in wastewater
treatment ensures the viability of the remediation process because the
loss of one microorganism can be compensated by the other microor-
ganisms integrating the consortia [3].
Several studies have reported the use of both native and articial
consortia in wastewater polishing (Table 6). For example, a non-native
consortium composed by Chlorella sp. and Scenedesmus sp. was effec-
tively applied in nitrogen and phosphorus removal from a primary-
treated municipal wastewater by Koreivien et al. [112]. These authors
reported total nitrogen (TN) and total phosphorus (TP) removal ef-
ciencies ranging between 88.696.4 and 99.799.9%, respectively. Addi-
tionally, the authors evaluated the potential of this consortium in CO2
mitigation and lipid production, concluding that the consortium was
able to accumulate about 0.651.37 g CO2 L 1 d1 in their biomass.
However, no lipid accumulation was observed in the studied conditions.
Using a native microalgal consortium from a carpet mill industry efu-
ent for biofuel application, Chinnasamy et al. [113] have reported a
high biomass and lipid production potential (9.217.8 ton ha1 yr1
and 6.82%, respectively), as well as an almost complete removal of
NO3-N and PO4-P (removal efciencies ranged between 96.6 and
99.8%). In order to obtain a self-occulating culture, Renuka et al. [3]
have evaluated the potential of a non-native consortium (composed
by unicellular and lamentous microalgae) and two native consortia
(one composed by lamentous microalgae and the other composed by
unicellular microalgae) in the treatment of a primary-treated sewage,
reaching high nitrogen and phosphorus removal efciencies in all stud-
ied consortia: NO3-N removal efciencies ranged between 81.5 and
83.3%, NH4-N removal efciencies were about 100% and PO4-P removal
efciencies ranged between 94.9 and 97.8%.
4.2. Microalgal-bacterial consortia
Competitive and cooperative interactions between microalgae and
bacteria have already been reported in the literature [15,24,114,115].
Fig. 2 evidences the possible interactions established between these mi-
croorganisms. Regarding competitive interactions, both microalgae and
bacteria can have adverse effects on each other. Several studies have al-
ready reported the excretion of microalgal metabolites presenting a
bactericidal effect [115117]. For example, chlorellin presents
 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx 7

Table 6
Application of microalgal consortia in nitrogen and phosphorus removal from different wastewaters and respective removal efciencies.

Nitrogen Phosphorus

System and operation Removal Ci Ci

Microorganisms Waste stream mode time (d) (mg L1) %R (mg L1) %R Ref.

Microalgal consortium composed by Ulothrix zonata, Digested dairy Immobilized system 7 78 TN 62 7 TP 70 [104]
Ulothrix aequalis, Rhizoclonium hieroglyphicum and manure wastewater (biolm reactor),
Oedogonium sp. semi-continuous mode,
V = 220 L
Microalgal consortium composed by Ulothrix zonata, Undigested dairy Immobilized system 7 64 TN 60 14 TP 93 [104]
Ulothrix aequalis, Rhizoclonium hieroglyphicum and manure wastewater (biolm reactor),
Oedogonium sp. semi-continuous mode,
V = 220 L
Calothrix sp., Lyngbya sp., Ulothrix sp. and Chlorella sp. Primary-treated Closed suspended 14 83.7 83.3 3.1 PO4-P 97.7 [3]
sewage water system, batch mode, NO3-N 100
V = 0.8 L 21.1
NH4-N
Chlamydomonas reinhardtii, Scenedesmus rubescens and Primary-treated Closed suspended 514 52.898.7 41.2100 3.911.5 12.2100 [141]
Chlorella vulgaris municipal system, batch mode, TN TN PO4-P
wastewater V = 5L
Chlorella sp. and Scenedesmus sp. Primary-treated Closed suspended 21 14.156.5 88.696.4 1.56.1 99.799.9 [112]
municipal system, batch mode, TN TP
wastewater at V = 0.23 L
different
concentrations
Chlorella sp., Scenedesmus sp., Chlorococcum sp. and Primary-treated Closed suspended 14 83.7 81.5 3.1 PO4-P 94.9 [3]
Chroococcus sp. sewage water system, batch mode, NO3-N 100
V = 0.8 L 21.1
NH4-N
Chlorella sp., Scenedesmus spp. and Chlorella zongiensis Dairy wastewater Closed suspended 34 176.0 TN 87.091.0 39.6 TP 91.296.0 [107]
system, batch mode,
V = 0.4 L
Chlorella vulgaris and Planktothrix isothrix Municipal Closed suspended 9 79.3 43.981.5 7.5 PO4-P 98.4100 [87]
wastewater system, batch mode, NH4-N
V = 0.25 L
Chlorococcum sp., Scenedesmus sp., Chlorella sp. and Synthetic municipal Closed suspended 14 n.a. n.a. 10 PO4-P 80 [25]
Phaeodactylum tricornutum wastewater system, batch mode;
V = 0.4 L
Microalgal consortium composed by the families Primary-treated Closed suspended 7 23.3 100 1.7 TP 100 [138]
Chlorophyta, Cyanobacteria, Euglenozoa and municipal system, batch mode NH4-N
Ochrophyta wastewater V = 1L
Microalgal consortium mainly composed by the genera Dairy wastewater Closed suspended 15 16.330.5 96 1.82.6 99 [136]
Actinastrum, Scenedesmus, Chlorella, Spirogyra, Nitzschia, system, batch mode, NH4-N PO4-P
Micractinium, Golenkinia, Chlorococcum, Closterium and V = 1L
Euglena
Microalgal consortium mainly composed by the genera Municipal Closed suspended 24 39.0 8499 2.1 PO4-P 9399 [136]
Actinastrum, Scenedesmus, Chlorella, Spirogyra, Nitzschia, wastewater system, NH4-N
Micractinium, Golenkinia, Chlorococcum, Closterium and semi-continuous mode,
Euglena V = 1L
Microcystis aeruginosa, Scenedesmus quadricauda, Chlorella Primary-treated Open suspended 5 0.64 45.3 4.6 PO4-P 32.6 [139]
vulgaris and Euglena viridis municipal system, batch mode, NO3-N 58.1
wastewater V = 60 L 39.4
NH4-N
Carpet mill industry wastewater native microalgal Carpet mill industry Closed suspended 3 1.43.9 99.799.8 17.622.0 96.699.1 [113]
consortium wastewater system, batch mode, NO3-N PO4-P
V = 0.5 L
Phormidium sp., Limnothrix sp., Anabaena sp., Westiellopsis Primary-treated Closed suspended 14 83.7 83.3 3.1 PO4-P 97.8 [3]
sp., Fischerella sp. and Spirogyra sp. sewage water system, batch mode, NO3-N 100
V = 0.8 L 21.1
NH4-N
Rhodobacter sphaeroides and Chlorella sorokiniana Synthetic high Closed suspended 1 b0.1 100 b0.1 100 [142]
strength organic system, batch mode, NO3-N 100 PO4-P
wastewater V = 0.2 L 0.1
NH4-N
Synechocystis salina and Chlorella vulgaris Synthetic medium Closed suspended 7 45 NO3-N 84.5 10 PO4-P 85.9 [143]
system, batch mode,
V = 0.45 L
Synechocystis salina and Microcystis aeruginosa Synthetic medium Closed suspended 7 45 NO3-N 77.7 10 PO4-P 97.2 [143]
system, batch mode,
V = 0.45 L
Synechocystis salina and Pseudokirchneriella subcapitata Synthetic medium Closed suspended 7 45 NO3-N 72.0 10 PO4-P 91.8 [143]
system, batch mode,
V = 0.45 L

Ci - initial concentration (in mg L1); %R - removal efciency; V - working volume; NO3-N - nitrate-nitrogen (mg N L1); NH4-N - ammonium-nitrogen (mg N L1); TN - total nitrogen
(mg N L1); PO4-P - phosphate-phosphorus (mg P L1); TP - total phosphorus (mg P L1); n.a. - not applicable.

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
8 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx
Fig. 2. Cooperative () and competitive () interactions established between microalgae and bacteria (adapted from Muoz and Guieysse [15]).
bactericidal activity against Gram positive and Gram negative bacteria,
such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseu-
domonas aeruginosa [118]. Additionally, the increase in pH and temper-
ature resulting from the photosynthetic activity can have a detrimental
effect on the co-cultivated bacteria [15,24]. Similarly, it has already been
reported that bacteria can excrete metabolites presenting an algicidal
effect [115]. On the other hand, a symbiotic relationship may be found
in microalgal-bacterial interactions. During photosynthesis, microalgae
release organic compounds that can be used by bacteria as carbon and
energy source and O2 that is used for the oxidation of organic matter.
On the other hand, bacteria release the CO2 required for photosynthetic
reactions [27,89,105,119,120]. However, cooperative interactions be-
tween microalgae and bacteria are more complex than simple nutrients
exchange. Microalgae can serve as a habitat for bacteria, protecting
them from adverse environmental conditions [24] and can enhance bac-
terial growth through the release of extracellular metabolites. Mandal et
al. [121] have demonstrated that extracellular polymeric substances
produced by the microalga Amphidinium carterae have stimulated the
growth of the bacterium Bacillus pumilus. Likewise, bacteria are respon-
sible for the excretion of growth-promoting factors, such as vitamins
(e.g. biotin, thiamine and cobalamine) and siderophores (important
chelating agents for microalgal growth under iron-decient conditions)
[23]. By increasing microalgal growth, an increase in nutrients uptake
rates is also expected. For example, in the study performed by De-Ba-
shan et al. [122], co-cultivation of Azospirillum brasilense with C. vulgaris
and Chlorella sorokiniana in alginate beads enhanced microalgal growth
and improved nitrogen and phosphorus removal from a municipal
wastewater used as culture medium. Apart from being effective in nu-
trients removal, these systems can further improve current wastewater
treatment processes because [89]: (i) the costs associated to the oxy-
genation of activated sludge tanks can be signicantly reduced; and
(ii) the greenhouse effects associated to wastewater treatment plants
can be considered negligible, since the CO2 released by bacteria is con-
verted into organic matter by microalgae.
Table 7 presents an overview of different microalgal-bacterial con-
sortia that have been effectively applied in nitrogen, phosphorus and or-
ganic matter removal from different wastewater sources. Taking into
account the CO2/O2 exchanges observed in these consortia and the asso-
ciated benecial effects, the activated algae concept has emerged as an
attractive strategy for nutrients removal from wastewaters, combining
the secondary and tertiary treatment steps in a single process [123].
These systems have been rstly proposed by Oswald et al. [124] using
high rate algal ponds (HRAPs), paddlewheel mixed, shallow, raceway-
type ponds that are completely oxygenated [2,6,86,125,126]. Since this
rst report, HRAPs have been successfully applied around the world,
being able to treat different wastewater streams, such as agricultural,
domestic and industrial [2,125127]. Additionally, several authors
have reported the potential of these systems in biofuels production
[86,125,126,128]. In these reactors, microalgae live together with
Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
heterotrophic bacteria, promoting an effective removal of BOD, COD
and nutrients [2,125]. When treating a domestic wastewater using a
HRAP, Park and Craggs [126] have reported NH4-N and PO4-P removal
efciencies of about 90 and 70%, respectively. In this study, average
lipid content determined in microalgal biomass ranged between 20
and 25%, being 40% the maximum lipid content achieved (under nitro-
gen limiting conditions). Besides the advantages already reported for
HRAPs, these systems present some limitations regarding (i) light pen-
etration and mixing, which are responsible for an imbalance between
bacterial respiration and oxygen production by microalgae; (ii) the
presence of protozoa and predatory zooplankton; and (iii) the necessity
of a further harvesting step to separate small microalgae from the treat-
ed efuent. To overcome problems related to light penetration, mixing
and presence of predators, several studies have proposed wastewater
treatment using microalgal-bacterial consortia in closed PBRs. When
growing a microalgal-bacterial consortium composed by C. vulgaris
and primary-treated municipal wastewater native bacteria in tubular
PBRs processing a primary-treated municipal wastewater (NH4-N and
TP concentrations of 17207 and 1.419.5 mg L1, respectively), He et
al. [103] have demonstrated removal efciencies ranging between
30.9 and 100% for nitrogen and between 65 and 98% for phosphorus.
Alcntara et al. [129] have used a closed tank to evaluate the perfor-
mance of a microalgal consortium isolated from a HRAP treating diluted
vinasse and activated sludge native bacteria in nitrogen and organic
matter removal from a synthetic wastewater containing 120 mg L 1
of NH4-N and 200 mg L1 of total organic carbon (TOC). These authors
have reported NH4-N removal efciencies ranging between 75 and 96%
and TOC removal efciencies ranging between 86 and 90%. To avoid the
requirements for further harvesting of microalgal biomass, some studies
have reported the use of these consortia in immobilized growth sys-
tems. Posadas et al. [77] have used a biolm reactor to promote prima-
ry-treated domestic wastewater treatment (TN, PO4-P and TOC
concentrations of 91, 7 and 181 mg L 1, respectively) by a centrate
wastewater native microalgal-bacterial consortium. This study has re-
vealed TN, PO4-P and TOC removal efciencies of 70, 85 and 90%, respec-
tively. When growing C. sorokiniana and activated sludge native bacteria
in a tubular biolm PBR treating primary-treated piggery wastewater
containing 656 mg L 1 of NH4-N, 117 mg L 1 of PO4-P and
1247 mg L1 of TOC, De Godos et al. [89] have reported the following re-
moval efciencies: 94100% for NH4-N, 7090% for PO4-P and 45% for
TOC. Immobilization in solid carriers has also been reported in the liter-
ature. De-Bashan et al. [122] have used an immobilized culture of Chlo-
rella sp. and A. brasilense in alginate beads to treat a municipal
wastewater with the following composition (in mg L1): 0.14.3 NH4-
N, 45.2 NO3-N and 4.1 PO4-P. This study has resulted in an effective re-
moval of nitrogen (both NH4-N and NO3-N) and phosphorus, with re-
moval efciencies ranging between 92 and 100%. To reduce time and
costs associated to microalgal harvesting, some authors have also de-
scribed the use of articial consortia consisting of occulating
 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx 9

Table 7
Application of microalgal-bacterial consortia in nitrogen, phosphorus and carbon removal from different wastewaters and respective removal efciencies.

Nitrogen Phosphorus Carbon

System and Removal Ci

Microorganisms Waste stream operation mode time (d) Ci (mg L1) %R (mg L1) %R Ci (mg L1) %R Ref.

Centrate wastewater native Centrate wastewater Immobilized 10 666 TN 60 101 54 76 TOC 0 [77]
algal-bacterial consortium system (biolm PO4-P
reactor),
continuous
mode, V = 31 L
Centrate wastewater native Primary-treated Immobilized 10 91 TN 70 7 PO4-P 85 181 TOC 90 [77]
algal-bacterial consortium domestic wastewater system (biolm
reactor),
continuous
mode, V = 31 L
Centrate wastewater native Synthetic domestic Closed 7 50 NH4-N 99.8 7 PO4-P 99.8 300 TOC 100 [88]
algal-bacterial consortium wastewater suspended
system, batch
mode, V = 1.9 L
Chlorella sorokiniana and Potato-processing Open suspended 10 12.1 NH4-N 95 47.5 80.7 745 COD 86.1 [81]
activated sludge native bacteria wastewater system, PO4-P
semi-continuous
mode, V = 5 L
Chlorella sorokiniana and Primary-treated Immobilized 7 656 NH4-N 94100 117 7090 1247 TOC 45 [89]
activated sludge native bacteria piggery wastewater system (biolm PO4-P
reactor),
continuous
mode, V = 7.5 L
Chlorella sorokiniana and Primary-treated Open suspended 10 12.3 NH4-N 82.7 3.4 PO4-P 58.0 465 COD 58.1 [81]
activated sludge native bacteria piggery wastewater system,
semi-continuous
mode, V = 5 L
Chlorella sp. ES-13 and ES-30, Oil, metal and Immobilized 7 n.a. n.a. n.a. n.a. 1200 COD 51 [54]
Scenedesmus obliquus ES-55, chemical industry system (solid
several Stichococcus strains, wastewater carriers),
Phormidium sp. ES-90, V = 100 L,
Rhodococcus sp. Ac-1267,
Kibdelosporangium aridum 754
and two unidentied bacterial
strains isolated from a collector
pond
Chlorella sp., Pediastrum sp., Primary-treated Closed 0.67 44.3 TN 61.2 1.4 PO4-P 30.256.8 n.a. n.a. [130]
Phormidium sp., Scenedesmus municipal wastewater suspended
sp. and activated sludge native system,
bacteria semi-continuous
mode, V = 4 L
Chlorella spp. and Azospirillum Municipal wastewater Immobilized 6 45.2 15 4.1 PO4-P 36 n.a. n.a. [122]
brasilense system (alginate NO3-N 100
beads), batch 0.14.3
mode, V = 0.6 L NH4-N
Chlorella vulgaris and Azospirillum Synthetic wastewater Immobilized 2 0.8 NH4-N 93 4.9 75 n.a. n.a. [144]
brasilense system (alginate PO4-P
beads), batch
mode, V = 0.5 L
Chlorella vulgaris and Azospirillum Synthetic wastewater Immobilized 2 0.8 NH4-N 91 4.9 PO4-P 0 n.a. n.a. [144]
brasilense system (alginate
beads),
continuous
mode, V = 0.5 L
Chlorella vulgaris and Azospirillum Synthetic wastewater Immobilized 2 0.8 NH4-N 100 4.9 PO4-P 83 n.a. n.a. [144]
brasilense system (alginate
beads),
semi-continuous
mode, V = 0.5 L
Chlorella vulgaris and Bacillus Synthetic medium Closed 6 20 NH4-N 78 4 TP 92 n.a. n.a. [132]
licheniformis suspended
system, batch
mode, V = 0.1 L
Chlorella vulgaris and activated Synthetic wastewater Closed 2.74 75 TN 33.4365.96 n.a. n.a. 195 TOC 78.686.5 [133]
sludge native bacteria suspended
system,
semi-continuous
mode, V = 30 L
Chlorella vulgaris and brewery Brewery industry Closed 20 n.a. n.a. n.a. n.a. 21723846 1315 [134]
wastewater native wastewater suspended COD
microalgal-bacterial consortia system, batch
mode, V = 15 L

(continued on next page)

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10 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx

Table 7 (continued)

Nitrogen Phosphorus Carbon

System and Removal Ci

Microorganisms Waste stream operation mode time (d) Ci (mg L1) %R (mg L1) %R Ci (mg L1) %R Ref.

Chlorella vulgaris and Primary-treated Closed 820 17207 30.9100 1.419.6 6598 n.a. n.a. [103]
primary-treated municipal municipal wastewater suspended NH4-N TP
wastewater native bacteria system, batch
mode, V = 1.5 L
Microalgal consortium from a Synthetic wastewater Closed 220 120 NH4-N 7596 n.a. n.a. 200 TOC 8690 [129]
high rate algal pond treating suspended
diluted vinasse and activated system,
sludge native bacteria continuous
mode, V = 2.7 L
Microalgal consortium from a Domestic wastewater Open suspended 4 56 NH4-N 91.896.9 7.0 PO4-P 70.072.9 n.a. n.a. [126]
high rate algal pond treating system,
domestic wastewater and continuous
activated sludge native bacteria mode,
V = 8000 L
Municipal wastewater native Primary-treated Open suspended 814 50.1 TN 93.795.8 8.8 PO4-P 64.093.5 380 COD 91.296.2 [102]
microalgae and activated municipal wastewater system, batch
sludge native bacteria mode, V = 14 L
Piggery wastewater native Anaerobically-digested Open suspended 10 303.3494.9 64.493.9 n.a. n.a. n.a. n.a. [90]
algal-bacterial consortium piggery wastewater system, NH4-N
semi-continuous
mode, V = 3 L
Piggery wastewater native Fresh piggery Open suspended 10 323.9568.9 58.294.8 n.a. n.a. n.a. n.a. [90]
algal-bacterial consortium wastewater system, NH4-N
semi-continuous
mode, V = 3 L
Primary-treated wastewater Primary-treated Closed 8 14.618.9 100 3.84.9 54.572.6 36.963.6 75.2 [131]
native algal-bacterial municipal wastewater suspended NH4-N PO4-P TOC 98
consortium system, batch 103.0190.9
mode, V = 14 L COD
Scenedesmus sp. and anaerobic Starch wastewater Closed 5 3050 88.7 54 80.1 180010000 80.5 [145]
sludge native bacteria suspended NH4-N PO4-P COD
system, batch
mode,
V = 0.06 L

Ci - initial concentration (in mg L1); %R - removal efciency; V - working volume; NO3-N - nitrate-nitrogen (mg N L1); NH4-N - ammonium-nitrogen (mg N L1); TN - total nitrogen
(mg N L1); PO4-P - phosphate-phosphorus (mg P L1); TP - total phosphorus (mg P L1); TOC - total organic carbon (mg C L1); COD - chemical oxygen demand (mg O2 L1); n.a. - not
applicable.

microorganisms. Van Den Hende et al. [130] have reported the use of
microalgal-bacterial ocs, mainly composed by Chlorella sp., Pediastrum
sp., Phormidium sp., Scenedesmus sp. and activated sludge native bacte-
ria, to treat a primary-treated municipal wastewater. The results obtain-
ed in this study have shown that these ocs were able to remove 61.2%
of TN and 30.256.8% of PO4-P. Similarly, Su et al. [131] have grown a
settleable microalgal-bacterial consortium in a primary-treated munic-
ipal wastewater, reporting a complete removal of NH4-N, PO4-P removal
efciencies ranging between 54.5 and 72.6% and TOC and COD removal
efciencies of 75.2 and 98%, respectively. To further improve the ef-
ciency of these systems in nutrients removal, some authors have studied
the effect of culturing conditions, such as light, nutrients concentration,
pH and microalgal:bacterial ratio [88,102,132,133]. Liang et al. [132]
have studied the effect of pH in the performance of a microalgal-bacte-
rial consortium composed by C. vulgaris and Bacillus licheniformis in ni-
trogen and phosphorus removal from a synthetic medium, reaching
NH4-N removal efciencies of 78% and TP removal efciencies of 92%.
When evaluating the effect of algae:sludge ratios on nutrients removal
from a primary-treated municipal wastewater, Su et al. [102] have re-
ported TN removal efciencies ranging between 93.7 and 95.8%, PO4-P
removal efciencies ranging between 64.0 and 93.5% and COD removal
efciencies ranging between 91.2 and 96.2%.
5. Current challenges of microalgal consortia and research needs
Although several studies have successfully applied microalgal and
microalgal-bacterial consortia in nutrients removal from different
wastewater streams, further research on this eld is required for the op-
timization of culturing parameters in large scale units. First of all, the
continuous treatment of different pollutants require an adequate selec-
tion of the microorganisms integrating the consortia, as the pollutants
can decrease the photosynthetic activity, decreasing the effectiveness
of the treatment. Additionally, to obtain more effective consortia, able
to degrade specic pollutants, further studies regarding the engineering
of new consortia and the design of synthetic microbial communities, ar-
ticial consortia where at least one of the species involved has been ge-
netically engineered, should be performed to meet some challenges,
such as [23,106]: (i) maintenance of long-term homeostasis; (ii) main-
tenance of long-term effectiveness of the consortia, even when horizon-
tal gene transfer occurs; (iii) incorporation of stable changes into the
genomes of the microorganisms involved in the consortia; and (iv)
ne-tuning of the consortia performance. The stability of microbial con-
sortia relies on the following features: (i) communication within the
microbial consortia or between individuals (the exchange of metabo-
lites and molecular signals, such as quorum sensing signals); and (ii)
the division of labour. Accordingly, engineering of microalgal consortia
should allow re-introduction or elimination of microorganisms as
needed and the complete monitoring of the tasks developed within
the consortia. For example, metabolite proling contributes to the
understanding of the complex interactions established among the
microbial members and to the identication of economically important
metabolites. Metabolic proling, functional genomics and combinatori-
al biochemical approaches including metabolic engineering can be
applied for the development of microalgal consortia presenting high
ability for pollutants removal.
Moreover, most of the studies regarding microalgal consortia in nu-
trients removal have been performed in laboratory scale units, which
may not be representative of real conditions. Accordingly, important

Please cite this article as: A.L. Gonalves, et al., A review on the use of microalgal consortia for wastewater treatment, Algal Res. (2016), http://
dx.doi.org/10.1016/j.algal.2016.11.008
 A.L. Gonalves et al. / Algal Research xxx (2016) xxxxxx
advances include [27,119]: (i) the study of the effect of different envi-
ronmental conditions, such as light, nutrients availability, pH and tem-
perature, on the consortia behaviour; (ii) large scale outdoor
experiments; (iii) the complete understanding of the interactions
established between the microorganisms integrating the consortia,
which is not well documented for photosynthetic consortia; and (iv)
the development of reliable mathematical models that correctly de-
scribe the behaviour of these consortia, which may be very helpful in
process design and operational conditions determination.
In summary, microalgal consortia combining microorganisms with
different metabolic capacities, such as mixotrophy, specic metal bind-
ing abilities, afnities for different nitrogen sources and euryhalinity,
should be developed. Additionally, the complexity of the interactions
established between the microorganisms integrating the consortia
should be completely understood. This approach constitutes the rst
step towards the application of the ecological engineering concept to
microalgal remediation processes, resulting in the development of
more resilient and effective treatment systems.
6. Conclusions
Although microalgal cultivation for wastewater treatment purposes
has been extensively described in the literature, the use of microalgal
consortia in this eld is still in an early stage of knowledge, which is
mainly due to the wide variety of possible combinations that can be ob-
tained. This review focuses on the application of these consortia
(microalgal and microalgal-bacterial) in wastewater treatment from
different sources, highlighting the mechanisms involved in nutrients re-
moval (carbon, nitrogen and phosphorus) by microalgae and how the
interactions established between the microorganisms composing the
consortia can improve the removal process. Comparing the effective-
ness of nitrogen and phosphorus removal obtained with microalgal
and microalgal-bacterial consortia, this study has demonstrated that
similar removal efciencies have already been reported. However, the
interactions established in microalgal-bacterial consortia are well docu-
mented, whereas little is known about the interactions established be-
tween photosynthetic microorganisms. Additionally, microalgal-
bacterial consortia can be more advantageous than those exclusively
composed by photosynthetic microorganisms because they can be
used as a replacement of both secondary and tertiary treatment steps
of wastewater treatment, while microalgal consortia can only be ap-
plied in wastewater polishing (as a replacement of the tertiary treat-
ment step). Large scale application of these consortia requires,
however, further research in terms of consortia ecology and engineering
and optimization of cultivation conditions.
Acknowledgements
This work was nancially supported by: Project UID/EQU/00511/
2013-LEPABE (Laboratory for Process Engineering, Environment, Bio-
technology and Energy EQU/00511) by FEDER funds through
Programa Operacional Competitividade e Internacionalizao COM-
PETE2020 and by national funds through FCT - Fundao para a Cincia
e a Tecnologia; SFRH/BD/88799/2012 and SFRH/BPD/66721/2009.
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