Algal Research 46 (2020) 101775

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Review article

Microalgae-based nitrogen bioremediation T

a a,b,⁎
Hui Chen , Qiang Wang
a
State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng 475004, China
b
Innovation Academy for Seed Design, CAS, China

A R T I C LE I N FO A B S T R A C T

Keywords: Pollution poses an increasing threat to the environment and to human health. Nitrogen pollution is of great
Ammonia nitrogen concern, with nitrogen oxide (NOx) in the air and ammonia nitrogen in water being two major pollutants.
Bioremediation Culturing microalgae in NOx or wastewater with high concentrations of ammonia nitrogen would both reduce
Microalgae environmental pollution and provide a source of nitrogen for microalgal culture. However, for microalgae-based
Nitrogen pollutants
bioremediation to be feasible, many fundamental questions about algal biology must be addressed. This review
NOx
summarizes progress in microalgal biotransformation, outlines applications of this technology, and provides an
Photosynthesis
in-depth description of the current state of microalgae-based bioremediation of NOx or wastewater with high
concentrations of ammonia nitrogen. Furthermore, we present possible solutions to some of the obstacles that
must be overcome to realize the practical applications of microalgae-based bioremediation.

1. Introduction forms of N. Whereas inorganic N is mostly in the form of ammonia,

nitrite, and nitrate [9–11], organic N exists mostly as urea, protein, and
Since the end of the 20th century, environmental pollution has been amino acids [12]. Of all the forms of N in wastewater, ammonia N is
increasing at a tremendous rate, along with the rapid growth of de- considered to be the most important pollutant. Ammonia N usually
veloping economies, such as those of China and India. Pollution, refers to N in the form of free ammoniacal nitrogen (NH3) and am-
especially nitrogen (N) pollution of air and water, poses an existential monium ions (NH4+), and occurs at elevated levels in domestic was-
threat to the environment, human societies, and the economy [1,2]. tewater [13], industrial waste [14], and aquaculture waste [15].
Nitrogen is one of the most abundant elements on Earth, and many Even though excess N is a pollutant, in some contexts N is in short
different forms of nitrogen exist in the biosphere. Free N molecules in supply. With the rapid development of the chemical fertilizer, petro-
the atmosphere can be converted into combined N (i.e., N chemically leum, and chemical industries, the demand for ammonia N is in-
bonded to one or more elements other than N) via atmospheric, in- creasing. Biological N fixation, a process in which nitrogen-fixing mi-
dustrial, and biological N fixation [3–5]. croorganisms reduce N2 in the atmosphere to ammonia, is an important
Nitrogen oxides (NOx) are the major pollutants of air and exist in source of ammonia N [16–19]. However, the amount of ammonia N
many forms, such as N2O, NO, NO2, N2O3, N2O4, and N2O5. Among produced by biological N fixation far from meets the needs of agri-
these, NO accounts for 90–95% of the NOx present [6]. Accumulation culture and industry, and industrial N fixation has become a key solu-
of NOx in the atmosphere results in serious environmental problems, tion [20,21]. However, during the production, transportation, and
such as acid rain, photochemical smog, and haze, with great harm to utilization of ammonia N, a large amount of underutilized ammonia N
human health [7,8]. There are two main sources of NOx pollutants in is discharged into water, causing water pollution. In addition, ammonia
the atmosphere: natural emission and anthropogenic emission [6]. N is an important pollutant in aquaculture systems because natural
Naturally emitted NOx is stable and relatively balanced in the air, and is ammonia removal processes are too slow to remove all the ammonia
mainly derived from microbial activity, NH3 oxidation, volcanic erup- being produced [22].
tions, lightning, stratospheric photochemical processes, and photolysis Algae are a highly diverse group of photosynthetic organisms,
of NO2− in soil and in the ocean. Anthropogenic NOx is emitted by which use light energy to split water into molecular oxygen and protons
human and industrial activities. and to convert inorganic carbon (CO2) to sugar and, ultimately, biomass
The discharge of N in wastewater also threatens both ecological [23]. As the oldest category of photosynthetic organisms, microalgae
systems and public health. Wastewater contains organic and inorganic use sunlight, carbon, and nitrogen sources to grow rapidly [1]. As N

⁎
Corresponding author at: State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng 475004, China.
E-mail address: [email protected] (Q. Wang).

https://doi.org/10.1016/j.algal.2019.101775
Received 14 November 2019; Received in revised form 17 December 2019; Accepted 23 December 2019
Available online 30 December 2019
2211-9264/ © 2019 Elsevier B.V. All rights reserved.
H. Chen and Q. Wang Algal Research 46 (2020) 101775

constitutes 7–12% of the microalgal biomass, large-scale microalgal In the aquatic nitrogen cycle, the inputs of ammonia N are derived
culture requires a large nitrogen supply. The high concentration of NOx from organic waste and aquatic animals, whereas the only way to
in flue gas and ammonia N in wastewater can be used to support the consume ammonia N is by the growth of photosynthetic algae and
growth of microalgae, while simultaneously removing a serious pollu- aquatic plants. For microalgae, ammonia N is both a toxic substance
tant [24,25]. and an important nutrient. In addition to consuming ammonia N, algae
In general, NOx species processes consist of 95% NO and 5% NO2 produce oxygen through photosynthesis, which promotes oxidation of
[26]. NO in the gaseous phase diffuses directly into the algae cells and ammonia N [53,54]. Removal of ammonia N by algae can not only
is then oxidized to NO2− inside the cells, or it is first dissolved in the eliminate environmental pollution and reduce the toxicity of ammonia
aqueous phase and then oxidized to form NO2− [26]. NO2 dissolves N to humans and other organisms but also reduce energy waste and the
easily in water and forms NO2− and NO3−, both of which then access cost of producing algal products [24] (Table 1). Our previous work has
into the cytoplasm. In eukaryotic microalgae, NO3− must be first re- suggested that combining bioremediation of environmental pollutants
duced to NO2− by nitrate reductase (NR) in the cytoplasm, and NO2− with microalgal cultivation represents an effective method for
reduced by NR or directly from NO2− uptake is further reduced to achieving environmentally sustainable, cost-effective production pro-
NH4+ by nitrite reductase (NIR) in the chloroplast [27]. The NH4+ cesses [1,2,55–57].
derived from NO2− or directly from NH4+ uptake combines with 2- Studies on bio-DeNOx (or nitrite N removal) or ammonia N removal
ketoglutaric acid (2-OG) to form glutamic acid or other nitrogen-con- using microalgae have been limited, although research in this field is
taining substances, in a reaction catalyzed by glutamine synthetase- receiving increasing interest (Tables 2 and 3) [2,5]. Nitrogen trans-
glutamate synthase (GS-GOGAT) in the chloroplast [23]. Ammonia N is porters of microalgae are of special interest for understanding of ni-
also transported into mitochondria, where it combines with 2-OG to trogen assimilation. NO3− is taken into algal cells via the NO3−
produce glutamate in a reaction catalyzed by glutamate dehydrogenase transporter proteins, the high affinity nitrate transporters (NRT2) [58].
(GDH) [28]. While as the prokaryotic microorganism, both the assim- NRT2 is only localized at the plasma membrane, meaning that NO3−
ilation of nitrite and ammonia N in cyanobacteria take place in the assimilation takes place in cytoplasm. NRT2 are nitrate/nitrite bispe-
cytoplasm [29]. cific transporters, which involved in the entry of both nitrate and nitrite
To truly realize microalgal-based bioremediation of environmental into the cells [59,60]. In eukaryotic microalgae, there are two kinds of
N pollutants, many fundamental questions based on in-depth studies of nitrite transporters, NRT2 and nitrate assimilation related family
algal biology must be answered. Here, we review the current state of (NAR1) [61]. Various types of NAR1 transporters have been found in
efforts to combine microalgal cultivation with the bioremediation of eukaryotic microalgae. For example, in Chlamydomonas, NAR1.1,
NOx or high ammonia N. We also explore possible solutions to current NAR1.2, NAR1.5 are chloroplast nitrite transporter and NAR1.3,
bottlenecks in this process and discuss future prospects. NAR1.4 and NAR1.6 are believed to be plasmalemma transporters [62].
Nitrite assimilation in cyanobacteria cells takes place in the cytoplasm.
2. Microalgal cultivation: a new process for NOx (or nitrite N) and So far, two types of nitrite active-transport proteins have been found in
ammonia N removal cyanobacteria, the ATP-binding cassette (ABC)-type NRT and the ni-
trate-nitrite-porter family in Major Facilitator superfamily (MFS) (NRT2
Increasing NOx emissions from human sources have seriously af- homologs) [63]. In aqueous solution, ammonia N is usually mixed in
fected the environment and human life [30–32], and NOx removal NH3 and NH4+, but at nearly neutral pH only 1% is in the NH3 form.
(DeNOx) has become a focus of air pollution control. Selective catalytic NH4+ enters algal cells with the assistance of the specific ammonium
reduction and selective non-catalytic reduction are the most popular transporters (AMTs)/methyl-ammonium permease (MEP), whereas NH3
DeNOx methods [33]. However, given the high cost of ammonia and directly diffuses into algal cells without the assistance of a carrier
catalyst, the high flammability of the gas phase ammonia, and a DeNOx [29,59,64].
efficiency of 85%, these processes are expensive and have security and Furthermore, more researches have mainly focused on three as-
secondary pollution issues [34]. Moreover, these processes reduce NOx pects: the breeding of algal strains with high tolerance of N pollutants;
to the waste gas N2, which fails to achieve the purpose of utilizing NOx the effects of NOx (or nitrite N) or ammonia N on microalgae; and the
as a source of combined nitrogen. factors influencing the efficiency of N removal.
An alternative is to use biological processes, including nitrification
and denitrification by microalgae, for DeNOx, which has the advantage 3. Selecting algal strains for NOx (or nitrite N) or ammonia N
of low energy consumption, low cost, simplicity, and reduced secondary removal
pollution [2,35,36]. Microalgal DeNOx could not only treat flue gas to
reduce NOx emission, but also supply nitrogen for microalgal culture, 3.1. NOx (or nitrite N) removal
reducing the cultivation cost and providing high value-added products
such as pigments, proteins, and oils [26,37–39]. Compared with tra- The NOx tolerance of microalgae varies, and the main algal genera
ditional physical/chemical methods, microalgal bio-DeNOx avoids the used for NOx biotransformation are Chlorella, Chlamydomonas,
problems of expensive materials and easy corrosion, and has the ad- Scenedesmus, Synechococcus, and Spirulina [65]. Lee et al. [66] isolated a
vantages of less secondary pollution and of turning waste into treasure. Chlorella sp. KR-1 strain from water samples collected near a thermal
Excessive ammonia N in natural water and aquaculture water not power plant. This strain could withstand simulated flue gas containing
only causes eutrophication, resulting in black and fetid water, but also 15% CO2, 100 ppm SO2, and 100 ppm (v/v) NO. Chiu et al. [67]
has toxic effects on various human and animal organs, including the evaluated the efficiency of on-site elimination of NO and SO2 from flue
brain, kidneys, blood vessels, and lungs, as well as on photosynthetic gas by Chlorella. Some Chlorella strains could directly and efficiently
microorganisms and plants [40–47]. Therefore, ammonia N must be utilize the NOx and SO2 present in flue gases and cultures of a strain of
maintained within a certain safe range. Chlorella sp. MTF-7 aerated with flue gas could remove up to 70% of the
Common ammonia N removal methods include gas extraction [48], NO in the gas. Yoshihara et al. [68] used the marine microalgal strain
adsorption [49], precipitation [50], oxidation [51] and biological NOA-113 to remove NOx from a simulated flue gas containing 300 ppm
methods [52] (Table 1). Regardless of the removal method, ammonia N (v/v) NO and 15% (v/v) CO2 in N2. This alga removed 50% of the NOx
is considered a pollutant and a worthless waste. However, as ammonia from flue gas bubbled through the algal medium at a rate of
N is also an important nutrient, a method that could not only remove 150 mL min−1. Nagase et al. (2001) obtained an NO removal efficiency
ammonia N from the environment but also reuse it to generate eco- of approximately 50–60% over 15 days when a model flue gas
nomic value would be an ideal economic strategy for waste recycling. (100 ppm (v/v) NO and 15% (v/v) CO2 in N2) was supplied at a flow

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H. Chen and Q. Wang Algal Research 46 (2020) 101775

Table 1
Comparison of several common ammonium nitrogen removal methods.
Method Advantage Disadvantage

Physical method Air stripping High efficiency, flexible operation High cost, high energy consumption, secondary pollution
Break point chlorination Effective, low investment Secondary pollution
Chemical method Chemical precipitation High removal rate, the product can be used as slow- Secondary pollution
release fertilizer
Ion exchange Convenient operation, small area Pretreatment required, secondary pollution
Adsorption Simple operation, low energy consumption and low Limited material
cost, no secondary pollution
Biological method Common biological Effective, no secondary pollution Cumbersome process, high cost, large area; ammonia nitrogen is
denitrification converted into free N2, which wastes resources
Microalgal denitrification Effective, no secondary pollution, full utilization of Developing technology, practical applications currently unfeasible
nitrogen, stable, cost-effective

Table 2
DeNOx efficiency of several studies based on microalgal cultivation.
Strain NOx concentration (dosage) Flow rate Initial cell density DeNOx rate Reference

−1 −1
NOA-113 300 ppm 150 mL min 1.5 g L 50% [68]
(v/v) NO
−1 −1
Dunaliella 100 ppm (v/v) NO 150 mL min 0.7 g L 65% [72]
tertiolecta
Dunaliella salina 100 ppm (v/v) NO \ \ 96% [111]
Scenedesmus sp 300 ppm (v/v) NO 0.3 vvm \ 40%–45% [113]
Scenedesmus sp 300 ppm (v/v) NO \ \ 80%–85% [70]
Scenedesmus dimorphus 300 ppm (v/v) NO \ \ 0.457 g L−1 d−1 [69]
Chlorella sp. C2 88.25 mM (4059.5 ppm) NO2− \ 0.07 g L−1 60% [56]
Chlorella sp. C2 88.25 mM (4059.5 ppm) NO2− \ 0.35 g L−1 96% [57]
Chlorella sp. C2 16.77 mM (771.42 ppm) NO2− \ 0.07 g L−1 ~100% [55]

Table 3 it as an N source. In particular, Chlorella sp. C2 was shown to possess

Ammonia N removal efficiency of several study based on microalgal cultivation. excellent tolerance and acclimation, as evidenced by its ability to
Strain Working concentration Removal efficiency Reference
maintain and quickly recover photosynthetic efficiency under high-ni-
trite conditions [75].
Chlorella vulgaris ~125 mg L−1 3.61 mg L−1 d−1 [119]
Chlorella vulgaris 7.7 mg L−1 1.93 mg L−1 d−1 [118]
Chlamydomonas sp. ~100 mg L−1 3.77 mg L−1 d−1 [75]
SW15aRL
3.2. Ammonia N removal
Chlorella FACHB- 500 mg L−1 14.86 mg L−1 d−1 [105]
1563 For removal of ammonia N from wastewater by algal cultures, the
main genera used are Chlorella, Scenedesmus, Spirulina, Chlamydomonas,
and Nannochloropsis [65]. The growth of Chlorophyceae was affected by
rate of 150 mL min−1 to a culture of the unicellular microalga Duna- high levels of ammonia N, and the effect was even greater on other
liella tertiolecta. Jiang et al. [69] reported that Scenedesmus dimorphus unicellular algae [46].
maintained vigorous growth when a simulated flue gas containing 15% Chlorella shows good potential for the removal of ammonia N from
CO2, 400 ppm SO2, and 300 ppm (v/v) NO was injected into the wastewater. Gutierrez et al. [76] found that increases in ammonia N
medium, and that the culture biomass reached 3.20 g L−1 after 7 days concentration had little effect on the growth of Chlorella but sig-
of cultivation. nificantly inhibited the growth of Neochloris and Dunaliella. Ammonia
For removal by microalgae, NOx in flue gas is first dissolved in the N-tolerance, as exhibited by some Chlorella strains, is a precondition for
aqueous phase of the culture, after which the NOx is oxidized and as- the use of microalgae in wastewater treatment. In addition, Chlorella
similated by the algal cells. However, NO, the main component of NOx, can absorb and assimilate ammonia N for its own growth and meta-
is only sparingly soluble in water, and the dissolution of NO into the bolism [5,77], which is key to the removal of ammonia N.
culture medium is the rate-limiting step for NO removal [70]. In many The use of ammonia N from wastewater for Chlorella culture can not
studies, NOx or NO was pumped directly into the bioreactor for mi- only achieve the purpose of removing ammonia N, but also reduce the
croalgal culture, but the low NOx flux and massive cell density cost of Chlorella culture. The Chlorella biomass that is produced can be
[68,71–73] required for effective NOx removal make this approach eaten by fish and shrimp, reducing the cost of feed and fully realizing
impractical for industrial applications. It may be possible to improve the economic strategy of resource recycling in aquaculture. However,
the efficiency of NO or NOx removal by first dissolving the gases in excess ammonia N can be toxic to Chlorella, inhibiting cell growth and
water and then supplying the fixed nutrients of NO or NOx to algal cells photosynthesis, and even causing death [76]. Therefore, screening for
in controlled amounts. In general, the main component of NOx emitted Chlorella strains that can tolerate high levels of ammonia N is a key
from incineration processes is NO, which accounts for up to 95% of the obstacle limiting ammonia N removal based on Chlorella culture. Our
total [74]. Nitrite is the main component of NOx aqueous solutions, and previous analysis of the ammonia tolerance of 10 Chlorella strains re-
is commonly used to simulate dissolved NOx in water. Screening for vealed that the growth inhibition of Chlorella by ammonia N is strain-
nitrite-tolerant microalgal species, therefore, is a critical step in de- specific [5]. Strain FACHB-1563 had the highest tolerance to ammo-
veloping algal bio-DeNOx processes. We previously investigated the nium nitrogen among all strains tested, suggesting that it could po-
nitrite tolerance of different Chlorella strains and found that some (but tentially be used to remove excess ammonium nitrogen from waste-
not all) strains could acclimate to high concentrations of nitrite and use water in bioremediation efforts.

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H. Chen and Q. Wang Algal Research 46 (2020) 101775

4. Mechanisms of NOx (or nitrite N) and ammonia N toxicity in reaction center and donor side of PSII were subsequently inhibited [80].
microalgae
4.2. Mechanism of ammonia N toxicity
4.1. Mechanisms of NOx (or nitrite nitrogen) toxicity
The most significant effect of ammonia N on algae is growth in-
In most cases, nitrate is transported into algal cells and reduced to hibition, and the degree of growth inhibition varies among different
nitrite in the cytoplasm. The nitrite is then transported into the chlor- algal species. Under high concentrations of ammonia N, the growth of
oplast and further reduced to ammonia by nitrite reductase [56,78]. Chlorophyceae was the least affected, followed by Cyanophyceae,
NOx and nitrite N are toxic to microalgae, and are detoxified inside the Diatomophyceae, Raphidophyceae, Prymnesiophyceae, and
chloroplast, explaining why photosynthesis is particularly sensitive to Dinophyceae [46]. Among the Chlorophyceae, different species showed
these chemicals [79,80]. Therefore, to use microalgae for bio-DeNOx, different degrees of growth inhibition under the same ammonia N
we need a deep understanding of the toxic effects of NOx and nitrite N concentrations. Gutierrez et al. [76] evaluated the effects of different
on microalgae. concentration gradients of ammonia N on the growth of four green
As the main component of NOx is NO, nitrite is the main component algae: Neochloris oleoabundans, Dunaliella tertiolecta, Chlorella sor-
of NOx fixed salt with NaOH solution and is both toxic and beneficial to okiniana, and Nannochloropsis oculate. The growth of Chlorella sor-
microalgae [56,80]. Nitrite, which can be directly absorbed and utilized okiniana was barely affected by the increase of ammonia N concentra-
by microalgae as a nutrient form of nitrogen, is the intermediate pro- tion, but the growth of Neochloris oleoabundans and Dunaliella salina was
duct of the inorganic nitrogen assimilation process and plays an im- significantly inhibited. At nontoxic levels, the amount of ammonia N is
portant role in microalgal metabolism [81]. Yang et al. [82] found that a key factor affecting algal growth. Yuan et al. [100] analyzed the ef-
Botryococcus braunii could use 5 mM nitrite as the only nitrogen source fects of ammonia N at various concentrations on the growth of Spirulina
in the medium for cell growth and propagation. However, excessive platensis, and concluded that ammonia N promotes the growth of algae
nitrite supply is toxic to microalgae, resulting in the inhibition of at low concentrations, but inhibits growth at high concentrations.
photosynthesis and cell growth and even in death [83]. The effect of ammonia N on the pH of the culture is also an im-
Nitrite assimilation takes place in the chloroplasts of eukaryotic portant factor affecting algal growth. NH3 and NH4+ can be present in a
algae. In addition, carbon and nitrogen metabolism consume organic solution simultaneously. The pKa (ion dissociation constant) of the
carbon from assimilated CO2 and energy from photosynthetic electron NH4+/NH3 buffer system is about 9.25 at 25 °C, meaning that at
transport, as well as other electron transport chains [23,84,85]. Nitrate pH < 9.25, the dominant form is NH4+, whereas at pH > 9.25, the
assimilation into amino acids by chloroplasts and cyanobacterial cells dominant form is NH3. The amount of NH3 at a specific concentration of
requires electrons from reduced ferredoxin (Fd), which is produced by ammonia N can be calculated using the equation NH3 (%) = 100 /
photosynthetic electron transport, and carbon scaffolds in the form of (1 + 10 ∗ (pKa − pH)) [101], and is closely related to the pH of the
ketoacids produced by carbon metabolism [86]. Therefore, nitrite as- medium and increases with increasing pH. NH3 is thought to be a far
similation is closely tied to photosynthesis, consistent with the idea that more toxic form of ammonia N than NH4+ [46,102]. When increasing
the photosynthetic apparatus is the main site of nitrite action and that pH levels lead to increasing NH3 content, growth inhibition is ex-
the toxicity of excess nitrite to algae is mainly due to inhibition of acerbated accordingly. Kallqvist and Svenson [102] found that the
photosynthesis. Stemler and Murphy [87] treated chloroplasts with growth of Nephroselmis pyriformis halted at pH 8.7 and a total ammonia
different concentrations of nitrite and detected changes in photo- concentration of 10 μmol L−1. At the same concentration of ammonia N
synthetic oxygen production, concluding that high concentrations of and pH 7.5, the growth rate decreased by only about 30%. In addition,
nitrite could significantly reduce the photosynthetic activity of chlor- the number of algal cells in the initial inoculum could affect the growth
oplasts. Loranger and Carpentier [88] found that nitrite could prevent of an algal culture in the presence of ammonia N. In a study by Markou
electron transfer between the two photosynthetic reaction centers in et al. [103], higher initial densities of Arthrospira platensis could as-
chloroplasts. We showed that Synechocystis sp. PCC 6803 is sensitive to similate ammonia N faster, alleviating the toxicity of ammonia N and
nitrite and that photosynthetic activity decreased sharply in algal cells reducing its inhibitory effect on algal growth.
cultured with millimolar concentrations of nitrite. Ammonia N inhibits algal growth mainly because it inhibits pho-
The survival and growth of photosynthetic organisms could be in- tosynthesis. Markou et al. [104] found that ammonia N affected PSI,
fluenced by various environmental stresses [84,89,90], which usually PSII, the electron transport chain, and the oxygen-evolving center
exhibit adverse effects on photosynthetic system and photosynthesis (OEC) in Arthrospira and Chlorella. A study by Dai et al. [105] showed
[91–95]. Photosystem II (PSII) is more sensitive to environmental stress that the growth rate of cyanobacteria decreased to 65% of normal in
than photosystem I (PSI) and is one of the primary sites of stress-in- 10 mM ammonia N, due to damage to the OEC. A study of Synechocystis
duced damage to the photosynthetic apparatus. Decreases in PSII ac- sp. by Drath et al. [106] came to the same conclusion: ammonia N
tivity are often the main cause of decreases in photosynthetic activity mainly damaged the OEC manganese cluster, resulting in a decline in
[95]. Therefore, the effect of nitrite on PSII has been the focus of re- photosynthetic rate. Although the OEC is considered to be the main site
search on the response of the photosynthetic apparatus to nitrite stress. of ammonia N-induced damage to the photosynthetic apparatus, there
Earlier results showed that nitrite treatment inhibits electron transport is no clear explanation on how ammonia N damages the OEC. During
through PSII and stimulates electron flow through PSI by causing a photosynthesis, H2O is split by the OEC to produce O2. NH3, which is a
redistribution of the absorbed light energy away from PSII and toward structural analogue of H2O and an inhibitor of the water oxidation re-
PSI [96,97]. However, there are different opinions about what causes action of PSII, inhibits O2 production under alkaline conditions [107].
the decrease in PSII activity in the presence of excess nitrite. Sahay Hou et al. [108] speculated that NH3 might replace an oxygen bridge in
et al. [97] proposed that the decreased quantum yield of PSII electron the manganese cluster or cause minor changes in the structure of the
transfer and decreased PSII activity under nitrite stress was caused by substrate water molecules in the OEC of PSII. Recently, our work
the transition of state 2. Using thermoluminescence [97], EPR [98,99], showed that PSII was damaged when exposed to high levels of am-
and other methods, nitrite was found to damage PSII at multiple sites, monium N, with the OEC as the primary site of damage, and that
and a high concentration nitrite inhibited both the donor side and the electron transport from QA− to QB was subsequently inhibited [5].
acceptor side of PSII. We previously concluded that the decrease of PSII In conclusion, although there are differences in the details, both the
electron transfer activity was caused by the transition of state 2 under nitrite and ammonia N can directly damage the photosynthetic appa-
nitrite stress, and that nitrite stress disrupted PSII activity by a stepwise ratus of microalgae, with subsequent effects on other physiological
mechanism in which the acceptor side was the initial target and the processes (e.g., cell growth), and the differences of the primary targets

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H. Chen and Q. Wang Algal Research 46 (2020) 101775

Fig. 1. Working model for the competition between N as-

similation and PS II damage by nitrite and ammonium N in
eukaryotic microalgae (A) and cyanobacteria (B). In eu-
karyotic microalgae, the nitrite and ammonium damaging
pathways are indicated in green and red, respectively, solid
lines for primary and dotted lines for secondary inhibition to
PS II. Normally, absorbed and transported into the cytosol,
nitrate is reduced stepwisely to nitrite by nitrate reductase
(NR) in cytosol (in pink) and to ammonium by nitrite re-
ductase (NiR) in chloroplast (in pink), and then goes into the
nitrogen assimilation pathway (in blue). When nitrite is
transported into the chloroplast, it directly disrupts PSII (solid
green inhibition symbol) and the reaction center and donor
side of PSII are subsequently inhibited (dotted green inhibi-
tion symbol). Nitrite is concurrently reduced to ammonium by
NiR in the chloroplast (in pink). When ammonium nitrogen is
reduced from nitrite or transported from the extracellular
environment into the chloroplast, it is used as a (1) N source
that is assimilated into nontoxic organic nitrogen by GS-
GOGAT (blue) and (2) hazardous material that is toxic to
photosynthesis, which initially damages the OEC (solid red
inhibition symbol) and then blocks electron transport from
QA− to QB (dotted red inhibition symbol). The damaging ef-
fects on PSII are dependent on the assimilation rate, which is
further determined by GS-GOGAT activity (in purple). The
similarly counteraction interaction between N assimilation
and toxicity exists in cyanobacteria, but the assimilation of
nitrite and ammonia N take place in the cytoplasm. (For in-
terpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

between the nitrite and ammonium N could be decided by their redox PSII. Although the assimilation of nitrite and ammonia N in cyano-
states. Furthermore, because nitrite can be converted to ammonia in the bacteria take place in the cytoplasm, the similarly counteraction in-
chloroplast, excess nitrite can lead to accumulation of toxic levels of teraction between N assimilation and toxicity exists.
ammonia N if the rate of ammonia N production exceeds the cell's ca-
pacity to assimilate it. For this reason, highly efficient nitrogen assim-
5. Efficiency of NOx (or nitrite N) and ammonia N removal by
ilation can alleviate the toxic effects of nitrite and ammonia N on
microalgae
photosynthesis.
A working model for the interaction between N assimilation and
5.1. NOx (or nitrite N) removal
toxicity can be summarized as follows (Fig. 1): in eukaryotic micro-
algae, produced by nitrate reduction or transported from the extra-
Most studies into the use of microalgae for DeNOx have attempted
cellular environment into cytosol, nitrite is transported into the chlor-
to feed flue gas directly to microalgal cultures [109,110]. However, as
oplast and directly disrupts PSII activity by a stepwise mechanism in
progress has been slow and some key problems remain to be solved, this
which the acceptor side of PSII is initially targeted and then the reaction
approach has yet to be implemented on an industrial scale [1]. First, the
center and donor side are inhibited. Concurrently, nitrite is reduced to
solubility of NO in water is extremely low; a direct flue gas feed system
ammonium by chloroplast nitrite reductase. Ammonia N, produced by
cannot solve the problem that most of the NOx in flue gas is NO and
nitrite reduction or transported from the extracellular environment,
thus difficult to be absorbed. Second, most microalgal strains can barely
acts as both an N source, by being assimilated into nontoxic organic
tolerate the high levels of NOx in industrial flue gas. The toxic com-
nitrogen (L-Glu) in a reaction catalyzed by GS-GOGAT, and as a toxin
ponents, dust, and high temperature of flue gas frequently kill micro-
that damages the photosynthetic apparatus. Thus, the GS-GOGAT-cat-
algal cultures being used to absorb and fix the high concentrations of
alyzed assimilation of ammonia N counteracts its damaging effects on
NOx.

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H. Chen and Q. Wang Algal Research 46 (2020) 101775

Although the small amount of NO2 in NOx is readily dissolved in algae to remove ammonia N from water (Table 3). Scenedesmus sp. has
water and used by microalgae, the low solubility of NO in algal media is been used to remove ammonia N from anaerobic digestion wastewater
the limiting step for NOx removal by microalgae. Therefore, various and urban wastewater [116,117]. Chlorella has been used to remove the
strategies are used to improve the efficiency of NO dissolution. The low concentrations of ammonia N present in the wastewater of a
dissolution of NOx can be promoted by increasing the surface area of sewage treatment plant; the ammonia N content dropped by 50% after
the gas–liquid interface and extending the duration of exposure; 48 h of treatment and the removal rate of ammonia N was 1.93 mg L−1
common methods include reducing the bubble diameter and selecting a d−1 [118]. Furthermore, green algae were also shown to be effective at
vertical long tube bioreactor with a high length-to-diameter ratio. removing high concentrations of ammonia N. Chlorella completely re-
Nagase et al. [111] improved the efficiency of gas–liquid mass transfer moved ammonia N with an initial concentration of ~125 mg L−1, with
by adopting a countercurrent airlift reactor; when 100 ppm NO was an average removal rate of 3.61 mg L−1 d−1 [119]. The Chlamydo-
pumped into a Dunaliella salina solution in the form of small bubbles, a monas isolated by Paskuliakova et al. [75] had a good removal effect on
NO removal rate of up to 96% was obtained. However, the low NOx flux landfill leachate containing high concentrations of ammonia N
rate of this method results in a low Nox processing capacity that is (~100 mg L−1). After 24 days of treatment, the total ammonia N
inadequate for the treatment of large quantities of flue gas, restricting content decreased by > 50%, with a reduction rate of 3.77 mg L−1 d−1.
its industrial application. The addition of the chelating agent Fe(II) In our study, the Chlorella strain FACHB-1563 was screened and used to
EDTA can significantly improve the dissolution of NO in water [112]. In remove ammonia N with an initial concentration of 500 mg L−1 [5]. In
the study by Jin et al. [113], 300 ppm NO at a flow rate of 0.3 vvm was addition to being tolerant to high concentrations of ammonia N, this
used as a nitrogen source for a Scenedesmus culture. The NO removal strain exhibited a high assimilation capacity for ammonia N and grew
rate could be maintained at 40–45% for 12 days by adding 5 mM Fe(II) normally in cultures with high concentrations of ammonia N
EDTA. Santiago et al. (2010) cultured Scenedesmus in an airlift photo- (500 mg L−1); an average removal rate of 14.86 mg L−1 d−1 was
reactor with Fe(II)EDTA, and obtained an NO removal rate of 80–85%. achieved, with the highest removal rate being 34.27 mg L−1 d−1. This
However, O2, which is continuously produced in algal culture systems work provides a basis for developing strategies for the in-situ pur-
due to photosynthesis, oxidizes Fe(II)EDTA to Fe(III)EDTA, which ification of ammonia N wastewater using microalgae.
cannot chelate NO. This, along with its relatively high cost, limits the Although several hurdles exist before algal-based N pollution bior-
use of Fe(II)EDTA to promote NO removal. emediation can be used in industrial settings [1,2,65], we have devel-
In addition to enhancing the dissolution of NO in water, the initial oped effective technical solutions for some of these obstacles based on
fixation of massive amounts of NO or Nox into water and then the research conducted in our laboratory in recent years [5,55–57]. And a
cultivation of algal cells using the fixed nutrients is possibly a more comprehensive microalgae photoautotrophic cultivation based cyclic
effective way to improve NO or Nox removal efficiency. Previously, we economical system was proposed. As shown in Fig. 2, beside produces
demonstrated that flue gas fixed salts from a caprolactam production carbon neutral energy, the biomass power plant also produces ash and
plant (Sinopec's Shijiazhuang Refining & Chemical Company) could be flue gas (with CO2 and NOx as major contaminants), which could be
used in an economically viable approach for algal biofuel-based bio- used as nutrient elements, carbon and nitrogen source for microalgae
DeNOx. Culture of Chlorella in the industrial flue gas salts resulted in biomass production. After the extraction of high value-added products
and a 60% nitrite removal efficiency and a lipid content of 33% of the and bio-lipids, the algae biomass residues could be further used either
algal dry cell weight [56]. Moreover, considering the vast amount of as feedstock for biomass power plant, or as feed additive for aqua-
NOx-containing flue gases produced and the relatively low photo- culture and poultry, and the nitrogen rich wastewater of the later could
autotrophic growth of the algae, the biological DeNOx of industrial flue be further used as nitrogen source for algae cultivation. Thus, by using
gas by mixotrophic cultivation of the oil-producing green algae Chlor- sunlight, the microalgal cultivation cored cyclic economical system
ella was evaluated, and an impressive DeNOx efficiency of over 96% generates extra high value-added products, carbon negative bioenergy,
was attained, along with a biomass productivity of 9.87 g L−1 d−1 and and food proteins, without additional wastes production.
a bio-lipid productivity of 1.83 g L−1 d−1, providing the first realistic
strategy for using algal biofuel-based bio-DeNOx for large-scale in- 6. Summary and outlook
dustrial applications in a limited land area [57]. Recently, we con-
firmed that the nutrient-rich ash and flue gas generated in biomass Given that the potentials and limitations of microalgae-based N
power plants could serve as a nutrient source for Chlorella sp. C2 cul- pollution bioremediation have been apparent for a long time, the cur-
tivation, and the cultures reduced the NOx present in the flue gas and rent level of research in the field is extremely promising. Great progress
sequestered CO2, with a DeNOx efficiency of~100% and a CO2 se- has been made in developing strategies to use microalgae for the
questration rate of 0.46 g L−1 d−1 [55]. bioremediation of N pollution, especially NOx and ammonia N, in air
and water; however, due to a poor understanding of basic algal biology
5.2. Ammonia N removal and technological shortcomings, large-scale microalgae-based bior-
emediation cannot yet be realized and much work remains to be done.
It is reported that microalgae prefer to take up the most reduced As microalgal utilization of NOx and ammonia N varies tre-
form of nitrogen and the order of preference for nitrogen utilization is mendously among algal strains and species, it is essential to collect,
NH4+ > NO2− > NO3− [25]. Florencio and Vega [114] established screen, and select suitable microalgal candidates for different applica-
that nitrate and nitrite are simultaneously utilized by Chlamydomonas tions. With the deep understanding of algal basic biology and the
reinhardtii in the absence of ammonia, but that ammonia is pre- continuous progress of biotechnology, genetic modification of micro-
ferentially utilized when ammonia is present. In addition, de Montaigu algae would be also a worthy process to obtain appropriate species for
et al. [115] showed that a gene (CYG56) coding for a NO-dependent microalgae-based N pollution bioremediation. Large-scale and longer-
guanylate cyclase mediated the negative signaling by ammonium on term production will help optimize the cultivation methods and im-
expression of NR in Chlamydomonas, which is another possible reason prove the economic viability of microalgal bioremediation of environ-
that the ammonia is preferentially utilized. mental pollutants. Efforts should focus on reducing the costs of culti-
Using algae to remove ammonia N takes advantage of the ability of vation and resolving the technical bottlenecks of large-scale
algae to assimilate ammonia N into organic N. This high ability to as- implementation of this bioremediation approach. Breakthroughs in
similate ammonia N limits the damaging effect of ammonia N on algal algae-based environmental applications are required to fully realize the
photosynthesis and growth and improves the removal efficiency of industrial potential of algal resources.
ammonia N. Moreover, many studies have assessed the ability of green Specifically, future efforts should aim to (1) increase funding for

6
H. Chen and Q. Wang Algal Research 46 (2020) 101775

Fig. 2. Microalgae photoautotrophic cultivation based cyclic economical system, everything beside the cycle are net output. By using sunlight, the microalgal
cultivation cored system generates additional high value-added products, carbon negative bioenergy, and food proteins, without extra wastes production.

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