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Ammonium Utilization in Microalgae: A Sustainable Method for Wastewater Treatment

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In plant cells, ammonium is considered the most convenient nitrogen source for cell metabolism. However, despite ammonium being the preferred N form for microalgae, at higher concentrations, it can be toxic, and can cause growth inhibition. Microalgae’s tolerance to ammonium depends on the species, with various taxa showing different thresholds of tolerability and symptoms of toxicity. In the environment, ammonium at high concentrations represents a dangerous pollutant. It can affect water quality, causing numerous environmental problems, including eutrophication of downstream waters. For this reason, it is important to treat wastewater and remove nutrients before discharging it into rivers, lakes, or seas. A valid and sustainable alternative to conventional treatments could be provided by microalgae, coupling the nutrient removal from wastewater with the production of valuable biomass. This review is focused on ammonium and its importance in algal nutrition, but also on its problematic presence in aquatic systems such as wastewaters. The aim of this work is to provide recent information on the exploitation of microalgae in ammonium removal and the role of ammonium in microalgae metabolism.
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sustainability
Review
Ammonium Utilization in Microalgae: A Sustainable Method
for Wastewater Treatment
Giovanna Salbitani * and Simona Carfagna


Citation: Salbitani, G.; Carfagna, S.
Ammonium Utilization in Microalgae:
A Sustainable Method for Wastewater
Treatment. Sustainability 2021,13, 956.
https://doi.org/10.3390/su13020956
Received: 9 December 2020
Accepted: 11 January 2021
Published: 19 January 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
Dipartimento di Biologia, Universitàdi Napoli Federico II, Via Cinthia 21, 80126 Napoli, Italy; simcarfa@unina.it
*Correspondence: giovanna.salbitani@unina.it
Abstract:
In plant cells, ammonium is considered the most convenient nitrogen source for cell
metabolism. However, despite ammonium being the preferred N form for microalgae, at higher
concentrations, it can be toxic, and can cause growth inhibition. Microalgae’s tolerance to ammonium
depends on the species, with various taxa showing different thresholds of tolerability and symptoms
of toxicity. In the environment, ammonium at high concentrations represents a dangerous pollutant.
It can affect water quality, causing numerous environmental problems, including eutrophication
of downstream waters. For this reason, it is important to treat wastewater and remove nutrients
before discharging it into rivers, lakes, or seas. A valid and sustainable alternative to conventional
treatments could be provided by microalgae, coupling the nutrient removal from wastewater with
the production of valuable biomass. This review is focused on ammonium and its importance in
algal nutrition, but also on its problematic presence in aquatic systems such as wastewaters. The
aim of this work is to provide recent information on the exploitation of microalgae in ammonium
removal and the role of ammonium in microalgae metabolism.
Keywords: ammonia; ammonium assimilation; extremophiles; microalgae; wastewater
1. Introduction
Microalgae are single-cell photosynthetic organisms, ranging in size from a few
µ
m
to a few hundred
µ
m; they are considered the greatest primary producer of any aquatic
ecosystem. Although over 300,000 microalgal species exist, only around 30,000 have been
studied or documented [
1
]. Phylogenetically, microalgae include many different groups,
existing in various aquatic and terrestrial habitats, and are tolerant of a wide range of
temperatures, salinities, pH values, and different light intensities. Therefore, they represent
a huge group of organisms that can live in an extensive range of environments.
Recently, microalgae have attracted considerable interest worldwide due to their
potential extensive applications in the renewable energy, biopharmaceutical, and nutraceu-
tical industries [
2
,
3
]. A wide spectrum of biologically active compounds has been found
in algal biomass; for example, proteins, polyunsaturated fatty acids (PUFAs), pigments,
vitamins and minerals, and extracellular compounds such as oligosaccharides [
4
7
]. In
particular, microalgae accumulate a high lipid content, normally ranging from 10% to 50%
of dry weight [
8
,
9
]; but in some genera, such as Botryococcus, the lipid content can reach 60%
to 90% of dry weight [
10
,
11
]. Due to their fast growth rate, these microorganisms can be
easily cultured in closed bioreactors or open systems and achieve high biomass yields [
12
],
and their cultivation does not compete for resources used in conventional agriculture [
13
].
Photosynthesis in microalgae is similar to that in higher plants, although characterized by
a higher yield compared to terrestrial crops, as it is more efficient in transforming solar
energy into chemical energy [
14
,
15
]. However, microalgal photosynthesis and growth can
be affected by several factors, including light supply, temperature, pH, inorganic carbon
availability, salinity, and nutrients [
15
18
]. Particularly, among nutrients, nitrogen (N) is
considered one of the most critical for plant-cell growth, since it is a constituent of proteins
such as peptides, enzymes, chlorophylls, and energy-transfer molecules [
16
,
19
]. In fact, N
Sustainability 2021,13, 956. https://doi.org/10.3390/su13020956 https://www.mdpi.com/journal/sustainability
Sustainability 2021,13, 956 2 of 17
is the second most abundant element in microalgal cells following carbon (C), ranging from
1% to 14% of their dry weight [
20
]. Due to nitrogen’s important roles in cell metabolism, its
restriction is often considered a limiting resource for microalgae, and in general for plant
growth. Microalgae can utilize different nitrogen forms such as nitrate, nitrite, ammonium,
or organic nitrogen (e.g., urea and amino acids). However, the favorable N-form not only
differs from species to species, but the diverse N-source also can influence the biochemical
composition of the algal cells in different ways [
21
,
22
]. In recent years, due to microalgae’s
ability to use inorganic and organic nitrogen for their growth, their use in treating wastew-
aters, particularly those rich in ammonium, has been studied [
23
]. Excess N discharge into
water can lead to eutrophication phenomena in natural aquatic environments, as well as a
decline in shellfish habitats and aquatic plant life [
24
]. The effect of using microalgae in
wastewater treatments has been studied by many researchers to make N removal and the
process of eliminating contaminants and pollutants more sustainable [
24
]. Moreover, when
exploiting their metabolic flexibility to grow photo-, hetero- or mixo-trophically, microalgae
represent an interesting system for treating a wide range of wastewaters [25].
Water is a very important resource for human development. It is been estimated that
a shortage of 40% of water resources could occur by 2030 [
26
]. Therefore, the sustainable
treatment of wastewater and water reutilization represent an important challenge glob-
ally. Ammonium is the one of the most common pollutants in wastewaters. Studies on
ammonium-containing wastewater are not limited to its removal, but are also centered
on nutrient recycling. The high resistance of some microalgae to high ammonium con-
centrations makes it possible to use these organisms for bioremediation and wastewater
treatment. Furthermore, wastewaters from industrial processes can be characterized not
only by high N concentration, but also by extreme pH and temperature; in these kinds
of effluents, extremophilic microalgae, which are able to grow well in intolerably hostile
or even lethal habitats, can be employed. The utilization of microalgae to remove the
ammonium content in wastewater, as well as their high-value biomass production, are
becoming attractive solutions.
In our review, we explore several issues related to the use of ammonium from microal-
gae and their sustainable utilization in wastewater treatments (WWT). In particular, the
aim of this study is to review recent advancements in microalgal application for ammo-
nium removal from wastewaters. We critically review ammonium recovery in wastewater,
especially its biochemical and biological significance in microalgal cultivation. This review
seeks to provide new knowledge on the role of microalgae in ammonium removal and the
utilization of this nutrient in algal metabolism.
2. Nitrogen Sources for Microalgae and Ammonium Utilization
Nitrogen is an essential component in many molecules of all living matter. In the envi-
ronment, N actively cycles among water, atmosphere, and soil in different concentrations
and forms, such as dinitrogen gas (N
2
), ammonium (NH
4+
), nitrate (NO
3
), nitrite (NO
2
),
and organic nitrogen (e.g., urea, amino acids, and peptides) [27].
Dinitrogen gas is the most abundant form of N on Earth (~78% in the atmosphere),
but it can only be used by a limited number of bacteria and archaea; therefore, it represents
an inaccessible source for microalgae and plants in general. The exploitable inorganic N
sources in the environment are NH
4+
, NO
3
, and NO
2
, which show a diverse concen-
tration based on different habitats [
28
]. In the environment, the most abundant N source
for plant cells is nitrate; however, microalgae are often able to utilize different nitrogen
sources based not only on its availability in the environment, but also based on what
species they are. In aerated soil, nitrate concentrations can be variable (from 10 to 100 mM),
but the ammonium concentration is generally quite low (<1 mg Kg
1
), because it is rapidly
converted by bacteria to nitrate [
29
]. In some acidic and/or anaerobic environments, the
ammonium can represent the dominant inorganic form of N [
30
]. In ocean waters, the
estimated concentration of nitrate is between 7–31
µ
M; of ammonium, 0.001–0.3
µ
M; and
of nitrite, about 0.006–0.1 µM [28].
Sustainability 2021,13, 956 3 of 17
In plant cell, the assimilation of inorganic nitrogen into amino acids and proteins
requires energy and organic skeletons; microalgae such as plants prefer NH
4+
because
the metabolic cost to reduce ammonium to organic matter is lower than the cost for
other nitrogen forms reduction [
27
]. By using ammonium, the microalgae avoid energy
consumption due to nitrate/nitrite reduction, but also due to nitrate reductase (NR) and
nitrite reductase (NiR) enzymes production [
31
,
32
]. In fact, in contrast to nitrate and
ammonium, subsequent to its transport into the cell, is directly incorporated into amino
acids via GS-GOGAT cycle by glutamine synthetase (GS)-glutamate synthase (GOGAT)
enzymes (Figure 1), and in some green algae, also under certain conditions, via the NADP-
glutamate dehydrogenase (GDH) pathway [32,33].
However, some microalgae, such as Botryococcus braunii and Dunaliella tertiolecta,
prefer nitrate as an inorganic N source, showing a reduced growth in the presence of
ammonium [22,34].
In addition, ammonium is crucial not only as nutrient, but also as an environmental
signal for cellular response [
35
,
36
]. In fact, in Chlorella vulgaris, the expression level of GS
was up-regulated 6.4-fold under 10 mg L
1
of ammonium compared to that in 4 mg L
1
,
confirming the key role of GS in ammonium assimilation [36].
Sustainability 2021, 13, x FOR PEER REVIEW 3 of 19
ammonium concentration is generally quite low (<1 mg Kg
1
), because it is rapidly
converted by bacteria to nitrate [29]. In some acidic and/or anaerobic environments, the
ammonium can represent the dominant inorganic form of N [30]. In ocean waters, the
estimated concentration of nitrate is between 7–31 µM; of ammonium, 0.001–0.3 µM; and
of nitrite, about 0.006–0.1 µM [28].
In plant cell, the assimilation of inorganic nitrogen into amino acids and proteins
requires energy and organic skeletons; microalgae such as plants prefer NH
4+
because the
metabolic cost to reduce ammonium to organic matter is lower than the cost for other
nitrogen forms reduction [27]. By using ammonium, the microalgae avoid energy
consumption due to nitrate/nitrite reduction, but also due to nitrate reductase (NR) and
nitrite reductase (NiR) enzymes production [31,32]. In fact, in contrast to nitrate and
ammonium, subsequent to its transport into the cell, is directly incorporated into amino
acids via GS-GOGAT cycle by glutamine synthetase (GS)-glutamate synthase (GOGAT)
enzymes (Figure 1), and in some green algae, also under certain conditions, via the NADP-
glutamate dehydrogenase (GDH) pathway [32,33].
However, some microalgae, such as Botryococcus braunii and Dunaliella tertiolecta,
prefer nitrate as an inorganic N source, showing a reduced growth in the presence of
ammonium [22,34].
In addition, ammonium is crucial not only as nutrient, but also as an environmental
signal for cellular response [35,36]. In fact, in Chlorella vulgaris, the expression level of GS
was up-regulated 6.4-fold under 10 mg L
1
of ammonium compared to that in 4 mg L
1
,
confirming the key role of GS in ammonium assimilation [36].
Figure 1. Ammonium and nitrate assimilation in microalgae. NR: Nitrate reductase; NiR: Nitrite reductase; GS: Glutamine
synthetase; GOGAT: Glutamate synthase; Glu: Glutamic acid; Gln: Glutamine.
According to Kronzucker et al. [37], there is a short-term inhibition effect of NH
4+
on
nitrate-uptake due to the direct consequences of ammonium on the plasma membrane;
this prompt effect is apparent within minutes of NH
4+
exposure. Inhibition of nitrate
uptake is a highly variable process, depending on the species, their physiological status,
and on environmental conditions [38]. In fact, for some species of phytoplankton,
ammonium concentrations between 100–300 nmol L
1
can be enough to totally suppress
nitrate uptake [39], but concentrations up to 1–2 µmol L
1
are sometimes necessary for the
same effect in other aquatic microorganisms [38]. It was reported that the inhibition of
Figure 1.
Ammonium and nitrate assimilation in microalgae. NR: Nitrate reductase; NiR: Nitrite reductase; GS: Glutamine
synthetase; GOGAT: Glutamate synthase; Glu: Glutamic acid; Gln: Glutamine.
According to Kronzucker et al. [
37
], there is a short-term inhibition effect of NH
4+
on
nitrate-uptake due to the direct consequences of ammonium on the plasma membrane; this
prompt effect is apparent within minutes of NH
4+
exposure. Inhibition of nitrate uptake
is a highly variable process, depending on the species, their physiological status, and on
environmental conditions [
38
]. In fact, for some species of phytoplankton, ammonium
concentrations between 100–300 nmol L
1
can be enough to totally suppress nitrate up-
take [
39
], but concentrations up to 1–2
µ
mol L
1
are sometimes necessary for the same
effect in other aquatic microorganisms [
38
]. It was reported that the inhibition of nitrate
transport is probably due to the accumulation of a product of ammonium assimilation
such as glutamine [
40
]. In cyanobacteria, ammonium availability results in an immediate
inhibition of nitrate uptake; in particular, the bispecific nitrate/nitrite transporter NRT
(ABC-type transporter NrtABCD) is repressed, as well as the NR and NiR proteins [
41
]. On
the contrary, in Chlamydomonas acidophila grown in a medium containing NH
4+
as unique
Sustainability 2021,13, 956 4 of 17
source of inorganic N, the NR activity was low, but not absent; therefore, NH
4+
replete
cells after N-starvation show an NR activity higher than that of NO3grown cells [32].
Although the uptake of ammonium is usually fast and preferred to other forms of
nitrogen, at important concentrations and prolonged exposure, it may be toxic for algal
cells and cause growth inhibition [
26
,
42
]. In the follow section, the toxic effect and the
tolerance to high ammonium concentration will be discussed.
3. The Equilibrium Ammonium/Ammonia and Effect on Microalgae
In the environment, ammonia represents a volatile molecule with very high solubility
(~35% w/wat 25
C), and for this reason, it is easily found as a liquid solution. In water,
the sum of NH
3
and NH
4+
represents the total ammonia nitrogen (TAN) that constitutes a
buffer system ammonia/ammonium, as explained by the following formula:
NH4++ OHNH3+ H2O
This equilibrium between ammonium (NH
4+
; ionized form) and ammonia (NH
3
;
unionized form) depends on some parameters; in fact, the two chemical forms are readily
interchangeable depending upon the pH, temperature, and salinity of water [20,43].
Being the ion dissociation constant (pKa) of the NH
4+
/NH
3
buffer system 9.26 at
25
C, when the pH of the medium is less than 9.26, hydrogen ions are incorporated into
ammonia to produce ammonium ions that become the dominant species in the medium
(Figure 2) [44]. Therefore, as pH rises, the ammonia concentration increases noticeably.
Sustainability 2021, 13, x FOR PEER REVIEW 4 of 19
nitrate transport is probably due to the accumulation of a product of ammonium
assimilation such as glutamine [40]. In cyanobacteria, ammonium availability results in
an immediate inhibition of nitrate uptake; in particular, the bispecific nitrate/nitrite
transporter NRT (ABC-type transporter NrtABCD) is repressed, as well as the NR and NiR
proteins [41]. On the contrary, in Chlamydomonas acidophila grown in a medium containing
NH4+ as unique source of inorganic N, the NR activity was low, but not absent; therefore,
NH4+ replete cells after N-starvation show an NR activity higher than that of NO3 grown
cells [32].
Although the uptake of ammonium is usually fast and preferred to other forms of
nitrogen, at important concentrations and prolonged exposure, it may be toxic for algal
cells and cause growth inhibition [26,42]. In the follow section, the toxic effect and the
tolerance to high ammonium concentration will be discussed.
3. The Equilibrium Ammonium/Ammonia and Effect on Microalgae
In the environment, ammonia represents a volatile molecule with very high solubility
(~35% w/w at 25 °C), and for this reason, it is easily found as a liquid solution. In water,
the sum of NH3 and NH4+ represents the total ammonia nitrogen (TAN) that constitutes a
buffer system ammonia/ammonium, as explained by the following formula:
NH4+ + OH NH3 + H2O
This equilibrium between ammonium (NH4+; ionized form) and ammonia (NH3;
unionized form) depends on some parameters; in fact, the two chemical forms are readily
interchangeable depending upon the pH, temperature, and salinity of water [20,43].
Being the ion dissociation constant (pKa) of the NH4+/NH3 buffer system 9.26 at 25
°C, when the pH of the medium is less than 9.26, hydrogen ions are incorporated into
ammonia to produce ammonium ions that become the dominant species in the medium
(Figure 2) [44]. Therefore, as pH rises, the ammonia concentration increases noticeably.
Figure 2. Influence of the pH on NH4+/NH3 dissociation equilibrium in water at 25 °C, and the optimum pH growth of
some microalgae.
Figure 2.
Influence of the pH on NH
4+
/NH
3
dissociation equilibrium in water at 25
C, and the optimum pH growth of
some microalgae.
In natural waters, ammonium is present in much greater concentrations than ammonia
due to the prevalence of circum-neutral pH. In fact, it was estimated that in seawater
(pH 8.00, 20
C), ~90% of TAN is present as ammonium ions [
45
]. According to Erikson [
46
],
the ratio NH
4+
/NH
3
increases 10-fold for each rise of pH unity and 2-fold for each 10
C
rise of temperature between 0–30 C.
Sustainability 2021,13, 956 5 of 17
The salinity also affects the NH
4+
/NH
3
equilibrium, but only with minor influence. In
fact, a raise in the ionic strength of the medium cause a very small decrease in the ammonia
content. An enhancement of salinity from 20 to 34% leads to a little diminution from 3.41
to 2.98% of ammonia [45,47].
Usually, cultivation systems of microalgae are enhanced with gaseous inorganic
carbon source (CO
2
) to improve the photosynthetic activity and the biomass production.
The pH of the medium and then the NH
4+
/NH
3
/availability can also depend on the
CO
2
assimilation rate of the algae [
44
]. If the CO
2
dissolution rate is superior to that of
assimilation, the continuous CO
2
insufflation leads to an HCO
3
and H
+
accumulation,
resulting in the acidification of the culture medium. On the contrary, in the presence of an
elevated microalgal assimilation rate, the CO
2
assimilation is not enough to satisfy the cells’
demand of inorganic carbon; in this case, the carbonate would be assimilated as a C-source
by the cell, which makes the K+/Na+accumulation and the medium alkalization [44].
In an alkaline environment, the ammonia represents the main form; it can diffuse
rapidly through membranes different to the ammonium that at low concentrations is taken
up by AMT high affinity transporters [
28
]. Therefore, the ammonia represents the most
toxic N form, having a direct impact on the photosynthetic apparatus of microalgae [42].
For plant cells, the NH
3
results are dangerous because it is unchargedand lipid soluble,
and thus passes through biological membranes more easily than the ionized form NH
4+
[
48
].
The toxicity of ammonia in autotrophic microalgae at first occurs through disrupting the
thylakoid transmembrane proton gradient [
42
,
49
,
50
]. Ammonia, diffusing through cell
membranes, affects not only the
pH component of the thylakoid proton gradient, but also
brings oxidative stress. When the NH
3
increases, it crosses membranes, leading to enhanced
flux through the chloroplast membrane into the thylakoid lumen [
49
]. Once in the thylakoid
lumen, NH
3
in the acidic environment is converted to NH
4+
, thereby decreasing the
transmembrane proton gradient needed to power ATP to ADP conversion [
49
]. Moreover,
according to Markou et al. [
50
], NH
4+
could affect the Oxygen Evolving Complex (OEC)
by shifting a water ligand to the outer Mn cluster of the OEC.
Wang et al. [
44
] proposed a model of ammonium nitrogen competition between N
assimilation and PSII damage. According to this model, when the adsorbed ammonium
was transported into the chloroplast, it could be taken as an inorganic N-source and assimi-
lated by the GS-GOGAT cycle and/or as a dangerous molecule, which primarily damages
the OEC and afterward blocks electron transport from Q
A
(primary quinone) to Q
B
(sec-
ondary quinone). Therefore, the negative effects on PSII are dependent on the assimilation
rate, which is further up to the GS-GOGAT cycle activity [
44
]. Ammonia compromises the
enzymatic activity of some proteins and the lipid peroxidation in membranes, and induces
a general cell-level disorder [51].
In ammonium tolerance, the GS/GOGAT activity plays an important role. In Chlorella
strains with high performance of the GS/GOGAT cycle, the NH
4+
is rapidly converted
into N-organic molecules, avoiding the ammonium accumulation and toxic effect for the
cells [44].
As stated before, the microalgae tolerance to NH
4+
/NH
3
varies from species to
species [
44
,
49
]. In fact, while Neochloris oleoabundans and Dunaliella tertiolecta showed an
inhibiting growth effect toward ammonia already at 2.3 and 3.3 mg L
1
, respectively,
Chlorella sorokiniana and Nannochloropsis oculata were largely unaffected by ammonia con-
centrations (0.2–16.7 mg L
1
) [
49
]. According to Zheng et al. [
52
], the threshold of NH
4+
toxicity in Chlorella vulgaris is around 110 mg L
1
, and Collos and Harrison [
45
] found
comparable results. At NH
4+
110 mg L
1
Chlorella cells maintain high F
v
/F
m
values and a
good photosynthetic efficiency [
52
]. A higher concentration of ammonium (220 mg L
1
)
corresponds in C. vulgaris to a low cell viability of 61% [52].
Katayama et al. [
13
] demonstrated the ammonium tolerance might be acquired as a
result of the acclimatization; in fact, two strains of Bacillariophyceae (Thalassiosira weissflogii
TRG10-p and TRG10-p105) acclimated up to 180 mg L
1
NH
4+
, which significantly sur-
passed the tolerance levels of 65 mg L
1
previously reported by Collos and Harrison [
45
].
Sustainability 2021,13, 956 6 of 17
According to Chuka-Ogwude [
53
], under high levels of ammonia, F
v
/F
m
values decreased
with the rising NH
3
-N concentration due to the disruption of the photosynthetic apparatus.
In acclimated microalgae, cells restore their homeostasis avoiding the toxic effects of the
stressor on photosystems.
The establishment of the inhibition of photosynthetic activities occurring in microalgae
is of particular importance in algae-based wastewater treatment processes. The resistance
or acclimation of some microalgae to high concentrations of ammonium makes it possible
to use these organisms for bioremediation and for treatment of wastewater usually rich in
ammonium/ammonia content.
4. Wastewater and Ammonium Content
Water is the most precious resource of our planet, and according to Adam et al. [
26
], it
could face a global shortage of 40% by 2030; for these reasons, the WWT represents a uni-
versal issue for a sustainable solution for water reutilization. Following the world’s growth,
the spread of wastewater production has become an important environmental problem.
Wastewaters must be treated for environmental protection before being discharged
into rivers, lakes, or seas [
54
]. The variability of the wastewaters (e.g., scales, contaminants,
pH, temperature) imposes different managements [
25
], but in general, wastewater may be
dealt with by 1. primary treatment: It removes sedimentable solid fractions; 2. secondary
treatment: It provides physical/chemical/biological processes, leading to the consumption
of organic matter and oxidation of the major nutrients; 3. tertiary treatment: It allows
disinfection and removes nitrogen, phosphorous, and traces of organic molecules [23].
Ammonia nitrogen represents the most common N-containing pollutants usually
found in the tertiary stage of conventional wastewater treatment. The removal of macronu-
trients as nitrogen represents one of the principal criteria for tertiary treatment [
23
]. A com-
plete and efficient tertiary process, aimed at removing TAN, but also phosphate in wastew-
ater, is usually more expansive than primary treatment [
55
]. The TAN concentrations vary
according to the nature of the wastewater, as shown in the Table 1. In industrial-based
wastewater, the ammonium concentrations may be in the range of
5–1000 mg L1[26].
Typically, activities such as food processing, rubber processing, textile and leather manufac-
turing, fertilizer plant, agricultural and zootechnical industries, etc., release high levels of
ammonium concentration [
56
]. Among the high-strength ammonium wastewaters, there is
the manure-free piggery wastewater, with a concentration of NH
4+
of ~220–2945 mg L
1
,
and wastewaters from coal gasification with 130–280 mg L
1
[
52
,
57
,
58
]. Concentrations
of NH
4+
/NH
3
up to 100 mg L
1
are generally derived from anaerobic digestion, when
ammonium is produced by degradation of the N-matter in the feedstock, primarily in the
form of proteins [
59
,
60
]. On the other hand, ammonium concentration in the municipal
wastewater ranges between 10–200 mg L1[26].
Table 1. NH3-NH4+concentration (mg L1) in different wastewater sources.
Wastewater Type NH3-NH4+Concentration (mg L1) Ref.
Municipal wastewater 27–100 [6163]
Domestic wastewater 39–60 [64,65]
Fish processing wastewater 8–42 [66]
Piggery wastewater 220–2945 [16,52,67,68]
Wool textile mill 54 [69]
Coal gasification 130–280 [57,58]
Paper mill 11 [70]
Soybean processing 90 [71]
Olive mill 530 [70]
Winery 110 [72]
Coke production 60 [73]
Dairy effluent 49 [74,75]
Sustainability 2021,13, 956 7 of 17
Ammonium pollution affects the water quality of water bodies causing numerous
environmental problems such as oxygen depletion, pH shift, cyanotoxin production, and
eutrophication of downstream waters: When N level is higher than 1.9 mg L
1
, the water
body is considered eutrophic [
76
,
77
]. In the last past decades, elevated N-nutrient contents
in the water layer have seriously compromised the biodiversity of freshwater ecosystems
worldwide [
78
]. In recent years, many sustainable technologies and methods are studied to
remove the TAN from water stream, leading its content under the recommended threshold
level from the World Health Organization (WHO) [56].
The TAN abatement in wastewater treatment is usually divided into different ap-
proaches: Physical (e.g., membrane separation), chemical (e.g., coagulation, solvent ex-
traction, ion exchange,), and/or biological [
23
,
56
,
79
]. The high effective cost and energy
consumption of technologies for one-step tertiary treatment of WWT represent a prob-
lem for industries and municipalities [
80
]. A valid alternative to conventional treatments
may be provided by microalgae, coupling the nutrient removal from wastewater with the
production of valuable biomass.
5. Microalgae in Wastewater Treatment for Ammonium Removal
In the last decade, microalgae have become important organisms for biological purifi-
cation of wastewater due their ability to utilize and/or accumulate nutrients, heavy metals,
and organic and inorganic substances in their cells/bodies. The use of microalgae for WWT
requires a minimum of mechanical equipment and reasonably little energy consumption
for their operations [
81
,
82
]. In wastewaters, N concentration must be reduced to acceptable
limits (generally <10–15 mg L
1
depending on discharge point, population, and region
regulation as described by regulation 91/271/CEE) before being released into the water
body [
83
]. Many studies demonstrated that microalgae have a great potential for N removal
and reported successful cultivations [
13
,
23
25
,
52
,
80
]. Green microalgae (Chlorophyceae)
represent the more exploited algae in WWT (Table 2).
It was calculated that for 1-ton microalgal production, about 40–100 Kg of inorganic N-
compound and 11–13 ML/Ha/year of water are required. It was also estimated that about
2500 m
3
of wastewater to obtain 1-ton of microalgal biomass could be treated [
80
]. There-
fore, at the same time, the utilization of wastewater for algal cultivation could allow the
removal nutrients from effluents and reduce the use of precious freshwater. According to
Yang and colleagues [
84
], using wastewater for microalgal cultivation would reduce ~90%
of the water demand and eliminate the requirement for nutrients. Wastewater could afford
most essential resources for large-scale of microalgal cultivation by providing inorganic
nutrients and organic matter for mixo- or heterotrophic cultures. In autotrophic microalgae
cultivation, algae can give a further possibility of integrating the nutrient removal with
carbon capture: CO
2
can be incorporated into biomolecules as proteins, carbohydrates,
and lipids by photosynthetic reactions [
55
]. Therefore, phototrophic cultivations could
represent a single-step solution to reduce the CO
2
emission as well as the eutrophication
by nitrogen [76,85].
In a culture medium, an ammonium concentration higher than 100 mg L
1
inhibits
the photosynthesis in some microalgal species [
42
,
45
,
66
]. Recently, several studies have
been conducted to alleviate TAN toxicity and to optimize the cell ammonium assimilation
in microalgal cultures and wastewater treatment [
20
,
42
,
45
]. It was reported that a high
concentration of TAN could be mitigated by increasing the initial culture cells concentra-
tion, modulating light intensity, and/or monitoring the pH of their medium [
20
,
42
,
50
,
86
].
In addition, microalgal growth and biomass yield are less affected by high ammonium
concentration using a mixotrophic cultivation; in fact, mixotrophic cells can provide more
energy for fast ammonium assimilation, reducing ammonium inhibition and increasing
the microalgal biomass [42].
According to recent studies (Table 2), microalgal systems can efficiently treat many
kinds of ammonium-containing-wastewaters: Domestic and urban wastewaters, livestock
Sustainability 2021,13, 956 8 of 17
wastes, agro-industrial wastewater, piggery effluent, effluent from food processing factories,
and so on.
To optimize the nutrient removal, microalgae can be employed in WWT as monocul-
ture, consortia, or combined systems. Monoculture or consortia systems refer to systems
using only a single or a consortium of microalgae for nutrient removal without the support
of other organisms [
82
]. Otherwise, combined systems use both microalgae and bacte-
ria for an efficient removal of nutrients (such as ammonium) and organic matter [
82
].
An interesting symbiosis for ammonium abatement is represented by nitrifying bacteria
(e.g., Nitromonas and Nitrobacter) and microalgae. Bacteria oxidizes ammonium into nitrite
and then to nitrate, and from the other side, microalgae by photosynthesis supply O
2
for oxidation and utilize CO
2
, deriving from bacterial respiration, for the Calvin–Benson
cycle [82,87].
Definitively, microalgae exploited in WWT represent an extensive area of research and
development with enormous potential. Although many studies were conducted regarding
the possibility of using microalgae as nutrient removers from wastewater, today only a few
studies regard algal cultivation under continuous wastewater administration [
88
]. Wastew-
ater treatment is a global issue on which much research is still ongoing. In fact, freshwater
has become a limited resource in many areas of the world. Continuous cultivation of
microalgae with wastewater could not only provide a continuous supply of microalgae
biomass, but it would also represent a great sustainable process for recycling wastewater.
Sustainability 2021,13, 956 9 of 17
Table 2. An overview of recent WWT research on microalgae.
Class Species Strain Wastewater Type pH NH4+-N
(mg L1)
NH4+-N Removal
(%)
Removal Time
(Days) Ref.
Chlorophyceae Chlorella sorokiniana UTEX 2805 Synthetic wastewater 10 100 5 [89]
Chlorella sorokiniana UTEX 1230 Digested from cattle manure 893 75 25 [90]
Chlorella sorokiniana UTEX 2714 Digested from cattle manure 893 59 25 [90]
Chlorella sorokiniana CS–01 Digested from cattle manure 893 75 25 [90]
Chlorella sorokiniana
Potato processing wastewater
5.8 12 >95 30 [91]
Chlorella sorokiniana Secondary pig manure 7.5 12 83 30 [91]
Chlorella vulgaris FACHB–30 Piggery wastewater 6.3 220 50 7 [52]
Chlorella vulgaris Landfill leachate 7 760 70 30 [80]
Chlorella vulgaris Domestic wastewater 7.3–8.4 2.7–11 80–87 1 [92]
Chlorella vulgaris LEM 07 Domestic wastewater 7.2 13 [93]
Chlorella minutissima Domestic wastewater 7.2 13 [93]
Chlorella zofingiensis Piggery wastewater 6.2 65–80 4 [93]
Chlamydomonas
reinhardtii Landfill leachate 7 760 70 40 [80]
Coelastrum microporum IFA9 Municipal wastewater 7.3 [62]
Graesiella emersonii ATCC 13482 Synthetic wastewater 4–16 >99 18 [88]
Neochloris oleoabundans LEM 17 Domestic wastewater 7.2 13 [93]
Scenedesmus dimorphus UTEX 1237 Animal production operation 7 45 95 [94]
Scenedesmus sp Anaerobic digestate 8 50–260 60–100 7 [95]
Scenedesmus sp Fertilizer Plant Wastewater 7.3 27 93 10 [96]
Cyanidiophyceae
Galdieria sulphuraria CCMEE 5587.1 Urban wastewater 1–4 15–25 63–89 6 [97]
Galdieria sulphuraria CCMEE 5587.1 Primary wastewater effluent 2.5 40 88 7 [98]
Galdieria sulphuraria 074G Food waste hydrolysates 2 500 [99]
Cyanophyceae Nostoc sp. Food–industry wastewater 7.6–9.8 44 [100]
Arthrospira platensis Food-industry wastewater 8.6–8.9 38–61 [100]
Phormidium tergestinum Slaughterhouse wastewater 9.2 [101]
Porphyridium purpureum Food–industry wastewater 8.6–8.9 43–58 [100]
Synechoccus nidulans LEM06 Domestic wastewater 7.2 13 [93]
Thermosynechococcus CL–1 Anoxic swine wastewater 7.2 359 50–100 0.5 [102]
Thermosynechococcus CL–1 Aerobic swine wastewater 7.5 182 30–60 0.5 [102]
Trebouxiophyceae
Botryococcus braunii LEM 14 Domestic wastewater 7.2 13 [93]
Botryococcus braunii UTEX LB 572 Domestic effluent 6.4 8 62–63 60 [103]
Botryococcus braunii BL C116 Domestic effluent 6.4 8 61–65 60 [103]
Sustainability 2021,13, 956 10 of 17
6. Extremophilic Microalgae in WWT
Extremophile microalgae are organisms that are able to grow well in intolerably
hostile or even lethal habitats, and which could represent a good solution for treatment
of “extreme wastewaters”. They can grow well under extreme pH, temperatures, salinity,
light intensities, nutrients available, and heavy metal concentrations. However, pH and
temperature are the major constraints affecting NH
4+
/NH
3
equilibrium in wastewater,
and for this reason, only these two will be discussed below.
In specific cases, wastewaters from industrial processes are characterized by high TAN
concentration and extreme pH (4.00 > pH > 8.00) and/or temperature (10
C > C > 40
C) that
are not compatible with the metabolism of most microalgae species [
25
]. For example, wa-
ter draining from the sites of mines is frequently rich in sulphate (100 to > 5000 mg dm
3
),
and it is acid with pH < 4 [
104
]. These kinds of wastewaters are highly polluting and
toxic to most life forms; if untreated, they may devastate the streams and rivers into which
they flow [
104
]. Acid wastewaters are usually derived from ammunition industries/labs,
pharmaceutical industries, mining sites, steel industry, electroplating and phosphorous
industries and, in many cases, their drainage is treated so as to be neutralized before being
released into the environment [
105
,
106
]. The low pH wastewater causes numerous prob-
lems in the effluent treatment as well as in the water body in which it is discharged [
105
].
Furthermore, a highly acidic medium breaks down organic matter and eradicates microbial
organisms able to treat the water naturally [105].
In environments with low pH, the number of algae able to survive are limited com-
pared to a neutrophilic environment. Microalgae described to be metabolically active in
highly acidic environments include some Chlorophyta, such as
Chlamydomonas acidophila
and Dunaliella acidophila, Chrysophyta, such as Ochromonas sp., and Euglenophyta, such as
Euglena mutabilis [
107
]. Poliextremophiles Rhodophyta (Cyanidium caldarium,
Galdieria sulphuraria
,
Galdieria phlegrea, and Galdieria maxima), frequently encountered in acidic waters and in
geothermal areas of the world [
19
,
107
109
], can be employed in the treatment of these
“extreme wastewaters”.
Red microalgae of the genus Galdieria (Cyanidiaceae) represent a very interesting or-
ganism, growing in cryptoendolithic habitats with pH ranging 0.50–4.00 [
17
,
107
109
]. All
Galdieria species are able to grow in the dark (heterotrophically) or in the light (mixotroph-
ically) by using numerous carbon sources as organic substrates, with about 50 different
carbon sources such as sugars, sugar alcohols, tricarboxylic-acid-cycle intermediates, and
amino acids [
17
,
108
,
110
]. Galdieria sp., being acidophilic organisms, colonize habitats with
very low pH in which the more available nitrogen form is present as ammonium; for this
reason, these organisms are well adapted to utilize NH
4+
as a principal source of inorganic
nitrogen and to tolerate prolonged exposure to this nutrient. According to Henkanatte-
Gederaet et al. [
97
], Galdieria sulphuraria is able to grow well in filtered primary-settled
urban wastewater (pH 2.5), considerably reducing organic carbon (46–72%) and ammonia-
cal nitrogen (63–89%), and it is able to grow on primary effluents, showing a high nutrient
removal rate [98].
In WWT, another important parameter for NH
4+
/NH
3
equilibrium is the temperature.
The wastewater temperature can depend on the production process of derivation, but also
on the climate of the region in which it is produced. In fact, according to Bugajski et al. [
111
],
a significant correlation between the temperatures of wastewater with the air temperatures
exists. Therefore, in the coldest and hottest regions, the wastewater is strongly influenced
by seasonal and night/day temperatures.
The increase in the temperature of the wastewater causes a change in the solubility
of oxygen, an acceleration of the oxygen adsorption process, the rate of bacteria activity,
and of some chemical and biological processes [
112
,
113
]. In particular, the free ammonia
proportion at 25
C relative to the TAN is about two-fold compared with 15
C [
112
,
114
].
There are three types of microalgae concerning the supporting temperature (Figure 3):
1. Mesophiles: Living at temperatures ranging between 15–45
C (T
C optimum ~32.5
C);
2. thermophiles: Growing at temperatures ranging between 40 to 75
C; 3. psychrophiles:
Sustainability 2021,13, 956 11 of 17
Growing in temperatures ranging between 0 and 20
C. Thermophiles and psychrophiles
represent the extremophile organisms, which are not only able to tolerate prohibitive
conditions, but require them for their metabolism [113].
In a wastewater pond in India, where the temperature ranges between 37–43
C, two
green algae, Asterarcys quadricellulare and Chlorella sorokiniana, were isolated [
113
]. Most of
the reported strains of Chlorella grow in an optimum range between 25–32
C, however,
some strains, after acclimation, become thermotolerant and can tolerate temperatures of
40 C or higher [89,113].
Sustainability 2021, 13, x FOR PEER REVIEW 13 of 19
thermophiles: Growing at temperatures ranging between 40 to 75 °C; 3. psychrophiles:
Growing in temperatures ranging between 0 and 20 °C. Thermophiles and psychrophiles
represent the extremophile organisms, which are not only able to tolerate prohibitive
conditions, but require them for their metabolism [113].
Figure 3. Relation of temperature and growth rates for representative psychrophilic, mesophilic, and thermophilic
microalgae.
In a wastewater pond in India, where the temperature ranges between 37–43 °C, two
green algae, Asterarcys quadricellulare and Chlorella sorokiniana, were isolated [113]. Most of
the reported strains of Chlorella grow in an optimum range between 25–32 °C, however,
some strains, after acclimation, become thermotolerant and can tolerate temperatures of
40 °C or higher [89,113].
Another challenge is the treatment of low temperature wastewaters. In fact, a large
variety of wastewaters, including domestic sewage and industrial wastewater, such as
those from bottling, malting, brewery, and soft drink manufacturing plants, are
characterized by low temperatures, and their heating is required, thus raising the costs of
this waste treatment [115,116]. Systems based on psychrophilic microalgae able to operate
at low temperatures (10–20 °C) offer the possibility to reduce the energy costs.
Koliella antarctica is a genus of psychrophilic unicellular green alga belonging to the
class of Trebouxiophyceae [117]. Koliella cells can be grown in a laboratory at a
temperature as low as 2 °C, but can adapt to different physical environmental parameters
such as temperature (from 2 to 20 °C) and light (from 8 to 60 µmol photons m2 s1); they
represent an interesting potential biological system for treatment of low temperature
wastewater, such as water from fresh fruit processing industries [25,117–119].
The extremophiles have shown to be promising for WWT, and for bioremediation.
Many of industrial wastes have harsh conditions, which make extremophiles and
polyextremophiles a good choice for their treatment before releasing them into the
environment [120].
Figure 3.
Relation of temperature and growth rates for representative psychrophilic, mesophilic, and thermophilic microalgae.
Another challenge is the treatment of low temperature wastewaters. In fact, a large
variety of wastewaters, including domestic sewage and industrial wastewater, such as those
from bottling, malting, brewery, and soft drink manufacturing plants, are characterized
by low temperatures, and their heating is required, thus raising the costs of this waste
treatment [
115
,
116
]. Systems based on psychrophilic microalgae able to operate at low
temperatures (10–20 C) offer the possibility to reduce the energy costs.
Koliella antarctica is a genus of psychrophilic unicellular green alga belonging to the
class of Trebouxiophyceae [
117
]. Koliella cells can be grown in a laboratory at a temperature
as low as 2
C, but can adapt to different physical environmental parameters such as
temperature (from
2 to 20
C) and light (from 8 to 60
µ
mol photons m
2
s
1
); they
represent an interesting potential biological system for treatment of low temperature
wastewater, such as water from fresh fruit processing industries [25,117119].
The extremophiles have shown to be promising for WWT, and for bioremediation. Many
of industrial wastes have harsh conditions, which make extremophiles and polyextremophiles
a good choice for their treatment before releasing them into the environment [120].
7. High-Value Molecules from Microalgae Exploited in WWT
The integration of microalgae with WWT offers promising opportunities to reduce
the treatment costs, to recycle the nutrients, and to obtain sustainable useful bioproducts
(proteins, pigments, lipids, carbohydrates, biofuel, etc.) [
25
]. The employment of appropri-
Sustainability 2021,13, 956 12 of 17
ate microalgal species in WWT allows for the hyperaccumulation of valuable bioproducts
without compromising the cells’ growth and their biomass increase.
The microalgae Scenedesmus strain SDEC8, grown in anaerobically digested effluent
kitchen waste, can accumulate more biomass than Scenedesmus SDEC13 grown in the same
culture medium [
121
]. This result indicates the importance of a proper choice of the algal
species and of the strain for WWT aiming to biomass or biomolecules production. In fact,
the ability to grow in the various wastewaters varies from species to species, and it is surely
strain dependent.
Prospectively, to reduce the economic costs of biofuel from algae, wastewater has been
proposed as an alternative nutrient source for microalgae cultivation [
25
,
103
]. According to
Rinna et al. [
103
], Botryococcus braunii (strain LB572), grown in domestic wastewater effluent,
shows not only an efficient nitrogen consumption, but also an effective intracellular lipid
production and accumulation, in particular regarding saturated fatty acids. Growing algae
in a low-cost medium is necessary to diminish the cost of microalgal cultivation and to
make the biofuel production a more economic and environment-friendly process [
122
]. In
Chlorella vulgaris, the biomass and lipid contents are appreciably higher for cells cultivated
in urban wastewater than in the basal medium since the lipid productivity that results
is about 1.5 times higher in cells cultivated in wastewater [
122
]. In contrast to biomass
production and productivity, the highest lipid accumulation was reached in microalgae
such as Chlorella ellipsoidea and Scenedesmus sp., cultivated in domestic secondary effluent,
characterized by low N content [
8
,
123
,
124
]. In fact, in some species of microalgae, lipid
content can reach up to 80%, usually under N-deficiency conditions [
10
,
11
]. For this reason,
in wastewaters, lipid productivity can be enhanced by two-stage cultivation, where lipid
content increases during the second stage characterized by N starvation [125].
The algal biomass also constitutes a valuable source of pigments, mainly chlorophylls
and carotenoids, which have been correlated with several health benefits [
125
]. From
Phormidium autumnale, grown under heterotrophic cultivation in slaughterhouse waste,
carotenoids at industrial scale to 108-ton year are produced [
125
,
126
]. In Thermosynechococ-
cus sp., grown in aerobic treated swine wastewater, satisfactory phycobiliprotein and
carotenoid contents were obtained [102].
The cyanobacterium Arthrospira platensis and the Rhodophytes Porphyridium sp. and
Galdieria sp. are the principal producers of phycobiliproteins [
19
,
100
,
108
]. Phycobilipro-
teins, in particular phycocyanin, have high commercial value as antioxidant and anti-
inflammatory molecules and represent safer ingredients for food, nutraceutical, and phar-
maceutical purposes [
3
,
108
,
109
]. The production of these pigments from microalgae cul-
tivated in wastewater has not been significantly explored. According to Arashiro and
colleagues [
100
], the cultivations of Nostoc sp., Arthrospira platensis, and Porphyridium pur-
pureum in food-industry wastewater showed efficient treatment of the wastewater, reaching
high removal of nutrients and interesting phycobiliproteins accumulation in algal biomass.
Several microalgal species, such as Chlorella ssp., Arthrospira platensis,Scenedesmus sp.,
Botryococcus braunii, and many others, show high nutrient removal capacity in wastewater
and return valuable molecules [
100
,
103
,
123
,
124
]. The application of these strategies for
microalgae utilization in WWT needs further study, but opens great possibilities in the
ammonium mitigation from effluents and in sustainable biomolecules production.
8. Conclusions
An excess of ammonium in water bodies can lead to an eutrophication phenomenon
in natural environments. For this, WWT represent an important global issue. The modern
WWT technologies in use are considered efficient in processing, but they necessitate a lot
of energy and do not contemplate the recycling of useful nutrients such as ammonium.
Microalgae are photo-, mixo-, and heterotrophic organisms that can be used in WWT for
removal of the inorganic nitrogen and of other pollutants. To improve the conventional
WWT, the utilization of microalgae for nutrient removal could represent a sustainable
Sustainability 2021,13, 956 13 of 17
solution. In fact, being algae producers of biologically active compounds, their biomass
obtained by WWT can be exploited as biomolecule sources.
Further research needs to focus on continuous microalgae cultivation in wastewater
that could not only provide a continuous supply of biomass, but would also represent a
great sustainable process for recycling wastewater.
Author Contributions:
The authors contributed equally to this work. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest:
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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... In the wake of rapid urbanization, industrialization, and a growing global population, wastewater management has become a critical environmental concern [1,2]. The intricate challenge lies in the wide range of pollutants that wastewater carries, with ammonium being a particularly detrimental constituent [2]. ...
... In the wake of rapid urbanization, industrialization, and a growing global population, wastewater management has become a critical environmental concern [1,2]. The intricate challenge lies in the wide range of pollutants that wastewater carries, with ammonium being a particularly detrimental constituent [2]. Ammonium, often present in substantial concentrations in both municipal and industrial wastewater, can pose significant threats to aquatic ecosystems [3]. ...
... Consuming high amounts of ammonium causes corrosive damage to the mouth, throat, and stomach [2]. As such, it is crucial to develop effective strategies for the removal of ammonium from wastewater to mitigate these environmental and public health issues. ...
Article
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The removal of ammonium from wastewater is a crucial process to mitigate the potential environmental issues associated with high nitrogen content in the water bodies. Natural zeolite, particularly clinoptilolite, has shown promise as an adsorbent for this purpose due to its unique surface properties. While zeolite has been studied extensively in this context, the current work represents an exploration of natural zeolite from The Tanggamus District, Indonesia. The study evaluated the impact of the desilication process on the ammonium adsorption capacity of this natural zeolite. The zeolites were characterized using XRD, XRF, and SEM. Batch adsorption studies revealed that the modified zeolite showed promising ammonium adsorption performance up to 35.4 mg/g. The adsorption data fit well with the pseudo-second-order kinetics and Freundlich isotherm model, suggesting chemisorption as the predominant mechanism. The effect of temperature and thermodynamic parameters highlighted the exothermic nature of the adsorption process.
... Lo anterior se debe a que durante el crecimiento exponencial las células se duplican de manera constante respecto al tiempo y se encuentran en un estado fisiológico activo donde sintetizan los metabolitos estructurales y las enzimas necesarias para su reproducción (Salama et al., 2017). En esta fase, las microalgas pueden asimilar el nitrógeno (NO3 − , NO2 − , NH4 + y urea) y el fósforo de las aguas residuales para sintetizar proteínas, péptidos, enzimas, clorofilas, DNA, RNA, ADP, ATP, lípidos y membranas celulares (Salama et al., 2017;Zhu et al., 2019;Salbitani & Carfagna, 2021). En el citosol, las microalgas reducen el NO3 − en NO2 − con la enzima nitrato reductasa que trasfiere dos electrones donados por el NADH, posteriormente, dentro del cloroplasto, el NO2 − es reducido en NH4 + por la enzima nitrito reductasa mediante la transferencia de seis electrones donados por la ferredoxina y, finalmente, el NH4 + es integrado en aminoácidos vía glutamato sintasa (GOGAT)glutamina sintetasa (GS) (Salama et al., 2017;Kumar & Bera, 2020;Salbitani & Carfagna, 2021). ...
... En esta fase, las microalgas pueden asimilar el nitrógeno (NO3 − , NO2 − , NH4 + y urea) y el fósforo de las aguas residuales para sintetizar proteínas, péptidos, enzimas, clorofilas, DNA, RNA, ADP, ATP, lípidos y membranas celulares (Salama et al., 2017;Zhu et al., 2019;Salbitani & Carfagna, 2021). En el citosol, las microalgas reducen el NO3 − en NO2 − con la enzima nitrato reductasa que trasfiere dos electrones donados por el NADH, posteriormente, dentro del cloroplasto, el NO2 − es reducido en NH4 + por la enzima nitrito reductasa mediante la transferencia de seis electrones donados por la ferredoxina y, finalmente, el NH4 + es integrado en aminoácidos vía glutamato sintasa (GOGAT)glutamina sintetasa (GS) (Salama et al., 2017;Kumar & Bera, 2020;Salbitani & Carfagna, 2021). La urea es asimilada por las microalgas mediante un mecanismo de cotransporte con iones sodio, una vez dentro de la célula es desaminada en dos etapas por la enzima ureasa o la urea amidoliasa para generar NH4 + que entra posteriormente en la vía GOGAT-GS (Kumar & Bera, 2020) mientras que el PO4 −3 es integrado en compuestos orgánicos durante el proceso de fosforilación que involucra la producción de ATP a partir de ADP (Salama et al., 2017;Liu & Hong, 2021). ...
... Se ha reportado ampliamente que, entre las fuentes inorgánicas de nitrógeno, la mayoría de las microalgas prefiere el amonio y no usan otra disponible hasta que éste se haya agotado o esté a punto de agotarse debido que su asimilación no requiere de energía ni de enzimas para ser reducido, como en el caso del nitrato, el nitrito y la urea (Salbitani & Carfagna, 2021). Sin embargo, la asimilación de nitrógeno en las microalgas es un proceso complejo que no permite declarar la preferencia universal por el amonio ya que algunas como Botryococcus braunii y Dunaliella tertiolecta prefieren el nitrato sobre otras fuentes de nitrógeno y muestran poco crecimiento en presencia de amonio (Kumar & Bera, 2020;Salbitani & Carfagna, 2020). ...
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Microalgae remove nitrogen and phosphorus from water for their growth and biomass production, wich has several biotechnological applications. This study evaluated the growth, NO3−, NO2−, NH4+, urea, PO43− and total nitrogen removal capacity, as well as the biomass production and the CO2 fixation of two new microalgae isolates, Chlorella sp. and Quadrigula sp., from the coast of Chiapas, México. Chlorella sp. had a higher specific growth rate (0.74/day) and a shorter doubling time (0.92 days) than Quadrigula sp. Removal per unit volume (mg/L) of NO3−, NH4+ and total nitrogen by Chlorella sp. was higher (26.88%, 10.58% and 16.63%, respectively) than removal by Quadrigula sp. On the other hand, daily removal (mg/L/d) of NO3−; NH4+; NO2−; urea and total nitrogen by Chlorella sp. was also higher (88%, 33%, 89%,15% and 50%, respectively) than daily removal by Quadrigula sp. The biomass productivity (mg/L/d) and the CO2 fixation rate (mg/L/d) were similar among both microalgae. The results of this study suggest that Chlorella sp. and Quadrigula sp. have potential for nutrient removal, biomass production and CO2 fixation.
... Additionally, the high nitrogen substrate concentration (ammonium sulfate at 4360 mg/L, C/N = 5) did not inhibit the growth of E. gracilis ( Figure S4). The high ammonium toleration of E. gracilis further revealed the advantage of cultivation under an initial pH of 3.5 since the ratio of toxic NH 3 to NH 4 + decreased as the pH decreased [34]. The acidic tolerance of E. gracilis is crucial in achieving high protein content when using ammonium sulfate as a nitrogen source. ...
... The acidic tolerance of E. gracilis is crucial in achieving high protein content when using ammonium sulfate as a nitrogen source. In addition, these results also suggested the potential of E. gracilis for acidic industries wastewater treatment, such as pharmaceutical industries, mining sites, and ammunition industries where ammonium concentrations are typically high (5-1000 mg/L) [34]. source and C/N ratio for E. gracilis protein production. ...
... Additionally, the high nitrogen substrate concentration (ammonium sulfate at 4360 mg/L, C/N = 5) did not inhibit the growth of E. gracilis ( Figure S4). The high ammonium toleration of E. gracilis further revealed the advantage of cultivation under an initial pH of 3.5 since the ratio of toxic NH3 to NH4 + decreased as the pH decreased [34]. The acidic tolerance of E. gracilis is crucial in achieving high protein content when using ammonium sulfate as a nitrogen source. ...
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Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.
... Nutrient disturbance in water has received increasing concerns due to its negative impacts on aquatic ecosystems, such as eutrophicationassociated algal blooms (Glibert, 2017) and animal husbandry-associated water pollution (Salbitani and Carfagna, 2021). As one of the main components of marine biofilms, pennate diatoms could respond to the replenishment of limited nutrients by regulating chlorophyll fluorescence (Zhao et al., 2015), thus showing potential as indicators of nutrient disturbance in water. ...
... The added concentrations of ammonium ranged from 2 mM (28 mg/L ammonium-N) to 250 mM (2400 mg/L ammonium-N), resulting in final concentrations ranging from 4 μM (0.056 mg/L ammonium-N) to 500 μM (7 mg/L ammonium-N). The added concentrations of ammonium were similar to those found in wastewater sources, which ranged from 8 mg/L (fish processing wastewater) to 2945 mg/L (piggery wastewater) (Salbitani and Carfagna, 2021). The final concentrations of ammonium in our study were similar to those observed in coastal waters. ...
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The phenomenon of nutrient-induced fluorescence transients (NIFTs) in algae has shown potential in indicating nutrient disturbance in water. However, it has only been reported in green algae with a narrow detection range. Marine biofilms, a type of microorganism aggregate with significant application potential, have been less investigated in relation to their NIFTs phenomenon. In the present study, we conducted a bioassay lasting 36 h to test the utilization of different nutrients by diatom-bacteria marine biofilms through detecting the effective quantum yield [Y(II)]. We found that the biofilms selectively utilized nitrate, ammonium, inorganic and organic phosphate, while not utilizing glycine and urea. Among the nutrients that could be used by the biofilms, only ammonium induced a significant NIFTs phenomenon. We observed a linear relationship between fluorescence changes during the ammonium-induced NIFTs and ammonium concentrations in both nitrogen-limited and phosphorus-limited biofilm cultures. Notably, a final concentration of 500 μM ammonium triggered significant increases in fluorescence compared to lower concentrations. Our study suggests that nutrient-limited marine biofilms have potential to monitor ammonium disturbance in water, with the significant increases in biofilm fluorescence serving as an alarm for ammonium input.
... Diversification after aeration suggested a synergistic effect between the algal reactor and the chicken house environment. The appearance of organisms belonging to the genus of Chlorella after the aeration may explain how the microbial community adapts to the new environment, as organisms belonging to this genus have shown a great tolerance against NH 3 , present in the exhaust air ( Figure S2), and have previously been found in contaminated wastewater plants (Salbitani and Carfagna 2021;Wang et al. 2018). Moreover, organisms belonging to Stramenopiles (Fig. 4, light violet), which include diatoms, were apparently enriched by exposure to exhaust air from the chicken coop. ...
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Sustainable approaches to circular economy in animal agriculture are still poorly developed. Here, we report an approach to reduce gaseous emissions of CO2 and NH3 from animal housing while simultaneously using them to produce value-added biomass. To this end, a cone-shaped, helical photobioreactor was developed that can be integrated into animal housing by being freely suspended, thereby combining a small footprint with a physically robust design. The photobioreactor was coupled with the exhaust air of a chicken house to allow continuous cultivation of a mixed culture of Arthrospira spec. (Spirulina). Continuous quantification of CO2 and NH3 concentration showed that the coupled algae reactor effectively purifies the exhaust air from the chicken house while producing algal biomass. Typical production rates of greater than 0.3 g/l*day dry mass were obtained, and continuous operation was possible for several weeks. Morphological, biochemical, and genomic characterization of Spirulina cultures yielded insights into the dynamics and metabolic processes of the microbial community. We anticipate that further optimization of this approach will provide new opportunities for the generation of value-added products from gaseous CO2 and NH3 waste emissions, linking resource-efficient production of microalgae with simultaneous sequestration of animal emissions. Key points • Coupling a bioreactor with exhaust gases of chicken coop for production of biomass. • Spirulina mixed culture removes CO2 and NH3 from chicken house emissions. • High growth rates and biodiversity adaptation for nitrogen metabolism. Graphical abstract Towards a sustainable circular economy in livestock farming. The functional coupling of a helical tube photobioreactor with exhaust air from a chicken house enabled the efficient cultivation of Spirulina microalgae while simultaneously sequestering the animals’ CO2 and NH3 emissions.
... Ammonium nitrate is taken up more easily and at a higher rate by microalgae. Nitrates, on the other hand, are converted into ammonium inside the microalgae chloroplast, making ammonium a better option in terms of the energy needed for the nitrate-to-ammonium conversion [106,107]. However, microalgae exposure to high ammonium concentration can suppress nitrate uptake, possibly due to a negative feedback loop, which may be attributed to the high production of glutamine in the cell upon exposure to ammonium [108]. ...
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Identifying microalgae biodiversity is essential to unleashing the diverse potential applications for microalgae. The aim of the chapter is to define the various tools and methodologies possible for revealing microalgae diversity. Identifying microalgae from natural environments, hotspots, and extreme environments enables us to isolate naturally tolerant species that may thrive and exhibit unique characteristics. Identifying microalgae will enable researchers to uncover new applications for these versatile organisms, including biofuel production, wastewater treatment, and pharmaceutical development. Hence, it is crucial to define the different possible identification approaches, beginning with the sample collection protocols and extending to the identification methods employed to determine the diversity of microalgae in an ecosystem.
... At pH below 9.26, hydrogen ions combine with ammonia to produce ammonium ions, which become the dominant species in the water. 49 Consequently, as the pH increases, the concentration of ammonia increases significantly and promotes toxicity to microalgae. 50 The sensitivity of microalgae to NH 3 toxicity varies between different species. ...
Article
Here we comparatively assessed eight different microalgae for possible recycling and bioconversion of residual nutrients from hydroponic effluent (HE) into biochemical-rich microalgal biomass. Among the tested strains, S. obliquus sp. 1 could take up nitrate, phosphate and ammonia up to 75.9, 80.8, and 66.7%, respectively. Meanwhile, the nitrate, phosphate and ammonia uptake for other tested microalgae was in the range of 30.6–73.8%, 72.4–72.7%, and 6.6–54.6%, respectively. Further, the highest biomass production, Bx (g L−1), specific growth rate, μ (d−1), and photosynthetic efficiency, PE (mmol per photon m2), were observed for S. obliquus sp. 1 (Bx = 1.42, μ = 0.21, and PE = 22.17) followed by Chlorella sp. (B = 1.35, μ = 0.16, and PE = 16.08) and Chlorella sorokiniana (B = 1.25, μ = 0.15, and PE = 15.99). Moreover, all the tested strains were found to be rich in major biomolecules including carbohydrates (5.76–10.49% TS), lipids (17.78–36.67% TS), and proteins (36.13–50.13% TS). The multivariant correlation analyses revealed that the bioconversion of nutrient-rich HE into biomass was positively correlated with the growth rate, photosynthetic efficiency, and biomolecule accumulation (p < 0.01). Overall, we conclude that S. obliquus sp. 1 may serve as a potential microalga for HE recycling and resource recovery leading towards a wastewater-based circular bioeconomy.
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The unicellular red alga Galdieria sulphuraria is a polyextremophilic organism with a metabolic flexibility to grow autotrophically or heterotrophically. Galdieria can also produce and accumulate biotechnologically attractive products such as pigments (phycocyanin) and proteins. In this research we studied the effects of nitrogen starvation and its subsequent restoration on pigment and free amino acid contents both in photoautotrophic and heterotrophic cells. Following the nitrogen starvation, the levels of the primary photosynthetic pigments decreased both in autotrophic and heterotrophic cells, except for the chlorophyll a marginally diminished in heterotrophic cells. Ammonium supply to G. sulphuraria N starved cells caused a significant increase of total chlorophylls both in autotrophic and heterotrophic cells. It was observed how such increase was more rapid and marked in heterotrophic cells than in the autotrophic ones. Under N starvation, phycocyanin contents decreased in both autotrophic and in heterotrophic cells; however, after a time-lapse of 24 hours, they resulted significantly higher in heterotrophic cells. In Galdieria sulphuraria, like in other microalgae, free amino acid contents were profoundly dependent on nitrogen status of the cells but heterotrophic cells maintained much higher levels, especially of glutamate, respect to autotrophic ones. In general, cells grown in the presence and absence of light showed different responses toward N availability; in particular heterotrophic cells seemed to respond quicker to the ammonium restoration compared to autotrophic ones.
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The present work evaluated the optimum concentration of microalgal cells for domestic wastewater treatment in terms of removal in nutrients and physicochemical parameters. In the study, three different concentrations (20, 30, and 40%) of microalgae was considered at 8 hours and 24 hours of Hydraulic Retention time (HRT). Among the different microalgal concentrations studied 30% microalgae concentration gave maximum removal at both the HRTs. The maximum removal efficiency of phosphate, ammonia and COD for the non-filtered sample was 87.67, 96.88, and 80.39%, respectively, for filtered sample it was about 91.32, 100, and 83.64%, respectively at 8 hours HRT. However, at 24 hours HRT maximum removal efficiency observed was 97.92, 92.22, and 93.47% for ammonia, COD and phosphate respectively in case of non-filtered sample whereas in filtered samples maximum removal efficiency was 100, 94.44, and 95.51% for ammonia, COD and phosphate respectively. From the study, it was found that microalgae can effectively remove nutrients and organic contents to desirable limits even at a low HRT of 8 hours. At the urban sector, if microalgae are incorporated in a conventional wastewater treatment system will enhance the cost-effective efficiency by lowering the HRT and increasing the removal efficiency with footprints of sustainable treatment.
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Bicarbonate ions are the primary source of inorganic carbon for autotrophic organisms living in aquatic environments. In the present study, we evaluated the shortterm (hours) effects of sodium bicarbonate (NaHCO3) addition on the growth and photosynthetic efficiency of the green algae Chlorella sorokiniana (211/8k). Bicarbonate was added to nonaxenic cultures at concentrations of 1, 2, and 3 g L-1 leading to a significant increase in biomass especially at the highest salt concentration (3 g L-1) and also showing a bactericidal and bacteriostatic effect that helped to keep a reduced microbial load in the algal culture. Furthermore, bicarbonate stimulated the increase in cellular content of chlorophyll a, improving the photosynthetic performance of cells. Since microalgae of genus Chlorella spp. show great industrial potential for the production of biofuels, nutraceuticals, cosmetics, health, and dietary supplements and the use of bicarbonate as a source of inorganic carbon led to shortterm responses in Chlorella sorokiniana, this method represents a valid alternative not only to the insufflation of carbon dioxide for the intensive cultures but also for the production of potentially bioactive compounds in a short period.
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In this study, the performance of TCL-1 cultivation in swine wastewater was observed under various light intensity, treatment type of swine wastewater, and initial biomass concentration. Furthermore, pigments production (phycobiliprotein and carotenoid), was the main target in this study along with optimum extraction method. Under the cultivation in the anoxic treated swine wastewater (ATSW), highest biomass increment (1.001 ± 0.104 g/L) was achieved with 2 g/L initial biomass concentration and 1,000 µE/m2/s light intensity whereas cultivation in the anoxic and aerobic treated swine wastewater (AATSW) presented better performance on pigments production with the highest production in allophycocyanin which reached 12.07 ± 0.3% dwc. Extraction time and ultrasonication have significant influence on the phycobiliprotein extraction, yet different temperature and incubation time give similar extraction result for β-carotene. Carotenoids production with AATSW cultivation were two times higher than the cultivation in ATSW. However, ammonium-N degradation was performed better in the ATSW cultivation.
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We isolated fifty-two strains from the marine aquaculture ponds in Malaysia that were evaluated for their lipid production and ammonium tolerance and four isolates were selected as new ammonium tolerant microalgae with high-lipid production: TRG10-p102 Oocystis heteromucosa (Chlorophyceae); TRG10-p103 and TRG10-p105 Thalassiosira weissflogii (Bacillariophyceae); and TRG10-p201 Amphora coffeiformis (Bacillariophyceae). Eicosapentenoic acid (EPA) in three diatom strain was between 2.6 and 18.6 % of total fatty acids, which were higher than in O. heteromucosa. Only A. coffeiformi possessed arachidonic acid. Oocystis heteromucosa naturally grew at high ammonium concentrations (1.4–10 mM), whereas the growth of the other strains, T. weissflogii and A. coffeiformi, were visibly inhibited at high ammonium concentrations (>1.4 mM-NH4). However, two strains of T. weissflogii were able to grow at up to 10 mM-NH4 by gradually acclimating to higher ammonium concentrations. The ammonium tolerant strains, especially T. weissflogii which have high EPA contents, were identified as a valuable candidate for biomass production utilizing NH4-N media, such as ammonium-rich wastewater.
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Food waste constitutes a significant portion of waste in the world. Indeed, it is estimated that about one-third of edible human food is wasted globally. Anaerobic digestion has been identified as a promising technology for the treatment of food waste as it generates a significant amount of energy and can remove a substantial portion of the organics. However, this process has not been adequately applied due to technical and economic challenges. Most importantly, anaerobic digestion of food waste produces waste in the form of Anaerobic digestate food effluent (ADFE), with high amounts of nutrient such as ammonium (up to 3000 mg L⁻¹ NH3-N). It has been established that this effluent can be used as a substrate for the cultivation of microalgae allowing both a means of its treatment and its possible valorization. This paper reviews the anaerobic digestion of food waste, the composition of its digestate and trends in the treatment of ADFE with emphasis on treatment using microalgae. Potential microalgal cultivation methods applicable to the treatment of anaerobic digestate, especially ADFE, and possible optimization of the cultivation methods are also reviewed critically. Further, understanding of the cultivation of microalgae in ADFE is required to aid in better design of its treatment process and valorization to improve its economics.
Chapter
By having toxic effects on human health, oxyanion contamination in wastewater is considered a priority among environmental concerns to be resolved. Widespread applications of the oxyanions in various industries and metal mining activities in certain environments resulted in high concentrations of these materials especially effluent in their toxic forms. One of the acceptable methods considered by a number of researchers to reduce their toxicity is bioreduction of oxyanions by microorganisms which is considered as one of the bioremediation methods. However, the major problem for bioremediation by conventional methods is the extreme conditions of industrial effluents, such as high salinity, high or low pH, and high temperature. Nature itself has given the solution to this problem, i.e., extremophiles, microorganisms that survive in these harsh conditions. Due to their ability in reducing metalloids and detoxifying them, they are suitable candidates for biological treatments. Here, the efficacy of various extremophile groups in bioremediation of major oxyanions including arsenic, selenium, chromium, and tellurium is reviewed.