**3. Bioremediation processes involving haloarchaea species**

The severe environment in which haloarchaea can survive makes this archaea a good agent for bioremediation in water treatment processes and in saline and hypersaline environments contaminated with toxic compounds such as nitrate, nitrite and ammonia, chlorine compounds such as perchlorate and chlorate, hydrocarbons, or heavy metals. New advances in the understanding of the haloarchaea metabolism, biochemistry, and molecular biology suggest that general biochemical pathways related to nitrogen (Nitrogen cycle), metals (iron, mercury), hydrocarbons, or phenols can be used for bioremediation processes. With regard to nitrogen species, it is interesting to note that denitrification and nitrification have been described so far as powerful pathways to remove nitrogenous compounds from wastewater. In this context, several bacterial species are shown to be excellent models for removing nitrogenous com‐ pounds contained in wastewater. However, the use of archaea to remove nitrogen from wastewater has been poorly studied. Denitrification and anaerobic ammonium oxidation (anammox) carried out by archaea can be practically used as efficient pathways to remove nitrate, nitrite, or ammonium from soils or wastewater. The existence of anammox (anaerobic ammonium oxidation, a part of the nitrogen cycle) was at first hypothesized based on thermodynamic calculations, and the hypothesis was subsequently confirmed in a pilot denitrifying wastewater treatment plant. Moreover, recent studies have suggested that anammox species are present not only in wastewater but also in marine- and freshwater with limited oxygenation, including oceans, seas, estuaries, lakes, and rivers. For example, the *amoA* gene coding oxidizing enzyme of ammonia was found from Crenarchaeota isolated from suboxic zones of marine environment by metagenomics. These results indicate that the nitrifying archaea can provide sufficient amount of nitrite by anammox under oxygen-limiting conditions. Thus, application of anammox may offer an attractive bioremediation process to current wastewater treatment systems for the removal of ammonia-nitrogen. In order to design a cheap and efficient process to nitrogen loss, anammox reaction could be coupled to nitrifi‐ cation carried out by archaea.

#### **3.1. Halophilic archaea in bioremediation of hydrocarbons**

**2.3. Nitrogen metabolism**

28 Advances in Bioremediation of Wastewater and Polluted Soil

[39] and some thermophiles [40].

**2.4. Sulfur compounds metabolism**

Some archaea species have reductive pathways of nitrogen such as assimilatory pathway (nitrate assimilation and N2 fixation) and dissimilatory pathway (nitrate respiration and denitrification). Nitrogen metabolism is much less known in archaea than in bacteria. How‐ ever, in *Haloferax mediterranei* some of these pathways are well known, making it a good

Many archaea are able to reduce nitrate by assimilatory or respiratory pathways through enzymes such as nitrate and nitrite reductases. These enzymes are found in a variety of halophilic and hyperthermophilic archaea [29,30]. Moreover, denitrification has been descri‐ bed for several halophilic archaea, such as *Haloferax* and *Haloarcula* strains [31, 32], and extreme thermophilic archaea, such as *Ferroglobus placidus* [33] and *Pyrobaculum aerophilum* [34]. However, fixation of N2 in archaea is exclusive of methanogenic euryarchaeota [35]. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase are the major pathways for ammonium assimilation in archaea. For example, both pathways have been identified in *Hfx. mediterranei* [36-38]. Recently, anammox systems have also been described from haloarchaea

Many archaea can utilize sulfur compounds as electron donors or acceptors for energy production [41]. Aerobic sulfur oxidation is common in Crenarchaeota (mainly in the order *Sulfolobales*); while anaerobic reduction of S0 is a widespread ability in the Crenarchaeota and Euryarchaeota phyla [42]. Dissimilatory sulfate, sulfite, and thiosulfate reduction are present in some thermophile genera of Euryarchaeota and Crenarchaeota [43,44]. Anaerobic DMSO respiration has been found in some haloarchaea that can grow anaerobically using DMSO as

The severe environment in which haloarchaea can survive makes this archaea a good agent for bioremediation in water treatment processes and in saline and hypersaline environments contaminated with toxic compounds such as nitrate, nitrite and ammonia, chlorine compounds such as perchlorate and chlorate, hydrocarbons, or heavy metals. New advances in the understanding of the haloarchaea metabolism, biochemistry, and molecular biology suggest that general biochemical pathways related to nitrogen (Nitrogen cycle), metals (iron, mercury), hydrocarbons, or phenols can be used for bioremediation processes. With regard to nitrogen species, it is interesting to note that denitrification and nitrification have been described so far as powerful pathways to remove nitrogenous compounds from wastewater. In this context, several bacterial species are shown to be excellent models for removing nitrogenous com‐ pounds contained in wastewater. However, the use of archaea to remove nitrogen from wastewater has been poorly studied. Denitrification and anaerobic ammonium oxidation (anammox) carried out by archaea can be practically used as efficient pathways to remove

candidate microorganism for bioremediation approaches [26-28]..

electron acceptor, such as *Halobacterium* sp. strain NRC-1 [45].

**3. Bioremediation processes involving haloarchaea species**

Many hydrocarbon contaminated environments are characterized by low or high tempera‐ tures, acidic or alkaline pH, high salt concentrations, or high pressure. The studies on the characteristics and degradation of hydrocarbon under these conditions suggest that the presence of extremophilic microorganisms living in extreme environments play an important role in the biological reaction and they could be useful for bioremediation. Halophilic extrem‐ ophiles seem to be very important to the bioremediation of oil-polluted salt marshes and treatment of industrial saline wastewaters. However, their full potential has not been suffi‐ ciently exploited [46]. Information on hydrocarbon degradation under high salt concentration is limited. Moreover, some authors reported the negative impact of increasing salinity on hydrocarbons biodegradation [47]. Most of these studies were performed using halophilic or halotolerant bacteria or bacterial consortia [48], but the potential alternative microorganisms are probably the extreme halophilic archaea.

Extreme halophilic microorganisms belonging to the Archaea Domain present diverse nutritional demand and metabolic pathways; for example, members of *Haloarcula* and *Haloferax* species use a variety of carbohydrates, organic acids as sole carbon and energy sources [49]. However, little is known about the ability of haloarchaea to grow in the presence of hydrocarbons as sole carbon and energy sources, although there is a lot of information related to physiology of microorganisms belonging to the *Halobacteriaceae* family.

#### *3.1.1. Halophilic archaea in bioremediation of crude oil and aliphatic compounds*

Zvyagintseva et al. [50] have reported the ability of halophilic archaea to degrade crude oil in hypersaline environments. Significant amount of isoprenoid and *n*-alkane fractions in crude oil were degraded by halophilic-like isolates from the brines of the Kalamkass oil fields in Kazakhstan. Al-Mailem et al. [51] isolated four extreme *Haloferax* archaea strains from the hypersaline coastal area of the Arabian Gulf. They were identified as two *Haloferax* strains, *Halobacterium* sp. and *Halococcus sp.* These halophilic archaea can use crude oil as sole source of carbon and energy, and the growth was enhanced by increasing the NaCl concentration in the medium. The optimum salt concentration value for growth was between 3.5 and 4.5 M. They also examined the effect of illumination and casamino-acids enrichment on the biore‐ mediation of crude oil using hypersaline soil and red pond water samples from the supertidal ''sabkha'' coastal area south of Kuwait [52]. The results indicated that addition of casaminoacids and exposure to light enhanced the oil consumption. Additionally, the antibiotics contained in the medium inhibit the growth of most bacteria except archaeal. These results suggest that degradation of hydrocarbons are mainly performed by haloarchaea species.

Some other researchers have reported archaeal ability to metabolize aliphatic hydrocarbons. Table 1 shows the extreme halophilic archaea able to degrade hydrocarbons and their carbon source. Bertrand et al. [53] isolated a halophilic archaeon, strain EH4, from a salt marsh in France. This strain was originally assigned to *Halobacterium* based on its phenotypic and biochemical characteristics but later analysis of the 16S RNA of strain EH4 indicated that it was closely related (99%) to *Haloarcula vallismortis* [54]. This strain was able to metabolize saturated hydrocarbons (tetradecane, eicosane, hexadecane, heneicosane). The growth of EH4 in the medium containing eicosano was salt dependent, growth and degradation was maxi‐ mum at 20% salinity and nondetectable below 10% salinity. Kulichevskaya et al. [55] have reported the isolation of an archaeon, *Halobacterium* sp. from hypersaline oil-contaminated wastewater in Russia. This strain grew optimally at 15-32% NaCl and showed a high capacity to degrade C10-C30 *n*-alkanes in a medium containing 30% NaCl. The *Haloferax*, *Halobacteri‐ um,* and *Halococcus* strains isolated on the basis of crude oil bioremediation [51] also degraded n-alkanes and mono- and polyaromatic compounds.


\*Bertrand et al. [53] labeled this strain as EH4 and assigned it to *Halobacterium sp.* based on phenotypic features. Tapilatu et al., [54] reassigned it to *Haloarcula vallismortis* based on 16 rRNA

**Table 1.** Extreme halophilic archaea able to biodegrade or grow on aliphatic hydrocarbons

Recently, the effect of vitamin and organic nitrogen on hydrocarbon removal was assessed by using halophilic bacteria and archaea from the Arabian Gulf. Al-Mailen et al. [56] proved the hydrocarbon remediation potential of five archaea in their natural hypersaline environments and how this potential can be enhanced by the addition of certain vitamins to the cultures. The most effective vitamins were thiamine, pyridoxine, and vitamin B12. These results were obtained not only for individual microorganisms in pure cultures but also for microbial consortia. Therefore, the supplement of vitamins could be an effective practice to enhance bioremediation of oil-contaminated hypersaline environments [56]. Tapilatu et al. [54] have reported the isolation of four alkane-degrading halophilic archaeal strains: one (strain MSNC 2) was closely related to *Haloarcula* and the others (strains MSNC 4, MSNC 14, and MSNC 16) were identified as *Haloferax*. These strains could degrade 32-95% of heptadecane when they were cultured in the medium containing 0.5 g/l of heptadecane and 22.5% NaCl for 30 days at 40°C. The strain MSNC 14 was also able to degrade phenanthrene. Otherwise, surfactants and emulsifiers are used to solubilise and disperse hydrophobic compounds. Halophilic archaea could be employed to this purpose. Post and Al-Harjan reported that the ether-linked phytanyl membrane of *Halobacteriaceae*, which showed emulsification properties, was effective in enhancing the efficiency of oil recovery [57].

#### *3.1.2. Halophilic archaea in bioremediation of aromatic hydrocarbons*

the medium. The optimum salt concentration value for growth was between 3.5 and 4.5 M. They also examined the effect of illumination and casamino-acids enrichment on the biore‐ mediation of crude oil using hypersaline soil and red pond water samples from the supertidal ''sabkha'' coastal area south of Kuwait [52]. The results indicated that addition of casaminoacids and exposure to light enhanced the oil consumption. Additionally, the antibiotics contained in the medium inhibit the growth of most bacteria except archaeal. These results suggest that degradation of hydrocarbons are mainly performed by haloarchaea species.

Some other researchers have reported archaeal ability to metabolize aliphatic hydrocarbons. Table 1 shows the extreme halophilic archaea able to degrade hydrocarbons and their carbon source. Bertrand et al. [53] isolated a halophilic archaeon, strain EH4, from a salt marsh in France. This strain was originally assigned to *Halobacterium* based on its phenotypic and biochemical characteristics but later analysis of the 16S RNA of strain EH4 indicated that it was closely related (99%) to *Haloarcula vallismortis* [54]. This strain was able to metabolize saturated hydrocarbons (tetradecane, eicosane, hexadecane, heneicosane). The growth of EH4 in the medium containing eicosano was salt dependent, growth and degradation was maxi‐ mum at 20% salinity and nondetectable below 10% salinity. Kulichevskaya et al. [55] have reported the isolation of an archaeon, *Halobacterium* sp. from hypersaline oil-contaminated wastewater in Russia. This strain grew optimally at 15-32% NaCl and showed a high capacity to degrade C10-C30 *n*-alkanes in a medium containing 30% NaCl. The *Haloferax*, *Halobacteri‐ um,* and *Halococcus* strains isolated on the basis of crude oil bioremediation [51] also degraded

**MICROORGANISM HYDROCARBON REFERENCE**

*Halobacterium sp. n*-Alkane C10-C30 [55]

*Haloarcula sp. Haloferax sp.* Heptadecane [54]

**Table 1.** Extreme halophilic archaea able to biodegrade or grow on aliphatic hydrocarbons

\*Bertrand et al. [53] labeled this strain as EH4 and assigned it to *Halobacterium sp.* based on phenotypic features. Tapilatu

Recently, the effect of vitamin and organic nitrogen on hydrocarbon removal was assessed by using halophilic bacteria and archaea from the Arabian Gulf. Al-Mailen et al. [56] proved the hydrocarbon remediation potential of five archaea in their natural hypersaline environments and how this potential can be enhanced by the addition of certain vitamins to the cultures. The most effective vitamins were thiamine, pyridoxine, and vitamin B12. These results were

*n*-Alkane C10-C34 [51]

[53]

Hexadecane Eicosane Heneicosane

n-alkanes and mono- and polyaromatic compounds.

et al., [54] reassigned it to *Haloarcula vallismortis* based on 16 rRNA

Strain EH4\* Tetradecane

30 Advances in Bioremediation of Wastewater and Polluted Soil

*Haloferax sp*. *Halobacterium sp.*

*Halococcus sp.*

Studies on aromatic hydrocarbons in earlier times were carried out with microorganisms isolated from samples of diverse hypersaline environments. Table 2 shows the typical halophilic archaea which can degrade aromatic compounds. In 1990, Bertrand et al. [53] isolated from salt-marsh an extremely halophilic archaea able to biodegrade aromatic carbons such as acenaphthene, phenanthrene, and anthracene. Later, Emerson et al. [58] described that *Haloferax* strain D1227 could grow in the medium containing monoaromatic compounds such as benzoate, cinnamate, and phenylpropionate by mineralizing as carbon sources, showing the physiological diversity of this group of archaea.

4-Hydroxybenzoic acid is a contaminant in certain highly saline industrial effluents. Fairley et al. [59] examined the metabolism of 4-hydroxybenzoic acid by *Haloarcula sp*. strain D1. The 4 hydroxybenzoic acid was changed to gentisate in the initial ring-cleavage reaction by the strain, although protocatechuic acid, hydroquinone or catechol is produced in case of the common pathways in aerobic bacteria, fungi, and yeast. In order to isolate new halophilic archaea able to grow in aromatic compounds, Cuadros-Orellana et al. [60] chose five different and unrelated hypersaline sites, the Uyini Salar (Bolivia), solar salterns in Cahuil (Chile), solar salterns in Cabo Rojo (Puerto Rico), *sabkhas* (Saudi Arabia), and the Dead Sea (Jordan). In this study, fortyfour new halophilic archaea able to grow in 4-hydroxybenzoic acid as sole carbon and energy sources were isolated (Table 2). Taxonomic characterization of these microorganisms revealed that the isolates represent at least four different groups of haloarchaea. They concluded that the ability to metabolize 4-hydroxybenzoic acid is widespread in the *Halobacteriaceae* family, and thus, these haloarchaea microorganisms are excellent candidates to bioremediate aromatic compounds of hypersaline environments and treatment of saline effluents. These authors also determined biodegradation kinetics of strain L1 isolated from the Dead Sea [61], and suggested that the strain L1 could degrade benzoic acid more efficiently than *Haloferax sp* D1227 [58]. Moreover, features about the benzoic acid catabolism were found in the *Haloarcula* sp. L1. When the strain L1 was grown in the medium containing benzoic acid, gentisic acid was produced, which was not usual in other microorganisms. Therefore, gentisic acid is an intermediate in the degradation of benzoic acid, hydroxybenzoic acid, cinnamate, and phenylpropionate by the archaea *Haloferax sp.* D1227, and in the degradation of 4-hydroxi‐ benzoic acid by the *Haloarcula sp.* strain D1 [59].

In relation to metabolism of the aromatic compounds in haloarchaea, the gentisate-1,2 dioxygenase genes (*gdoA*), which correspond to the ring-cleavage enzyme of gentisic acid, of *Haloferax sp.* D1227 and *Haloarcula sp.* strain D1 were cloned. Surprisingly, the expression pattern of the genes is different: in *Haloarcula* sp. D1, *gdoA* is expressed in the presence of 4 hydroxybenzoate but not benzoate; however, *gdoA* is expressed in *Haloferax sp.* D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate, and phenyl-propionate. Moreover, genes of Co-A synthetize (*acdB*) subunit and CoA-thioesterase (*tieA*) also existed at the upstream of the *gdoA* gene. The pattern of these genes expression is also different between the two species, obtaining only expression of *acdB* and *tieA* in *Haloferax sp.* D1227 during growth on benzoate, cinnamate, and phenypropionate, but not on 3-hydroxybenzoate. This suggests that *acdB* and *tieA* are part of benzoate degradation pathway in *Haloferax sp.* D1227, while the *gdoA* genes encode part of a 4-hydroxybenzoate and 3- hydroxybenzoate pathways in *Haloarcula* sp. D1 and *Haloferax sp.* D1227, respectively [62].

During the last four years, the number of studies which describe the degradation of aromatic compounds by halophilic archaea have increased. As already mentioned, Tapilatu et al. [54] isolated four haloarchaea from hypersaline environment. The MSNC 14, one of the strains (Table 2), was able to degrade 43% of phenanthrene after 30 days of incubation, although the degradation of anthracene and dibenzothiophene was not detected. Al-Mailen et al. [51] isolated four strains (Table 2) from a hypersaline coastal area for the Arabian Gulf on a mineral salt medium containing crude oil vapor as unique source of carbon and energy. The four strains were able to biodegraded not only aliphatic hydrocarbons but also aromatic hydrocarbons after three weeks of incubation. In particular, *Halobacterium* and *Haloccocus* could grow in the presence of benzene, toluene, and p-hydroxybenzoic acid, and the two *Haloferax* strains could grow with toluene and phenanthrene, and one of them also with benzene, but both failed to grow on p-hydroxybenzoic acid. This study also revealed that the biodegradation rates increased in proportion to NaCl concentration in the medium, and thus supported the idea that extreme halophilic archaea are suitable biological material to bioremediate oil-polluted hypersaline environments. Bonfá et al. [63] revealed the usefulness of halophilic archaea in the bioremediation of wastewater of petroleum production, which contained high saline concen‐ tration and various aromatic acids and hydrocarbons. Aromatic compounds (p-hydroxyben‐ zoic acid, naphthalene, phenanthrene, and pyrene) could be also degraded by nine halophilic archaea (Table 2) isolated from Çamalti Saltern, Turkey [64]. This study broadens the under‐ standing of metabolism of aromatic compounds, and the activities of catechol 1,2 dioxygenase and protocatechuate 3,4 dioxygenase were identified as the enzymes involved in *ortho* cleavage pathway. *Ortho* cleavage pathway is widely distributed in soil bacteria and fungi, constituting the major pathway for aromatic compounds catabolism in these organisms. Therefore, these enzymes may be important to remove aromatic hydrocarbons.

These results suggest the following: *Halobacteriaceae* family, which can degrade aromatic compounds, exists widely, and can be used to degrade aromatic compounds in oil-polluted hypersaline environments [60]. Thus, the bioremediation process using those strains is promising, as the remediation processes using physical and chemical methods are complicated and expensive [65]. However, more precise understanding of the mechanism of carbon-cycling cleavage and their enzymes and genes may be necessary to achieve the bioremediation [66].

phenylpropionate by the archaea *Haloferax sp.* D1227, and in the degradation of 4-hydroxi‐

In relation to metabolism of the aromatic compounds in haloarchaea, the gentisate-1,2 dioxygenase genes (*gdoA*), which correspond to the ring-cleavage enzyme of gentisic acid, of *Haloferax sp.* D1227 and *Haloarcula sp.* strain D1 were cloned. Surprisingly, the expression pattern of the genes is different: in *Haloarcula* sp. D1, *gdoA* is expressed in the presence of 4 hydroxybenzoate but not benzoate; however, *gdoA* is expressed in *Haloferax sp.* D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate, and phenyl-propionate. Moreover, genes of Co-A synthetize (*acdB*) subunit and CoA-thioesterase (*tieA*) also existed at the upstream of the *gdoA* gene. The pattern of these genes expression is also different between the two species, obtaining only expression of *acdB* and *tieA* in *Haloferax sp.* D1227 during growth on benzoate, cinnamate, and phenypropionate, but not on 3-hydroxybenzoate. This suggests that *acdB* and *tieA* are part of benzoate degradation pathway in *Haloferax sp.* D1227, while the *gdoA* genes encode part of a 4-hydroxybenzoate and 3- hydroxybenzoate pathways in *Haloarcula* sp. D1

During the last four years, the number of studies which describe the degradation of aromatic compounds by halophilic archaea have increased. As already mentioned, Tapilatu et al. [54] isolated four haloarchaea from hypersaline environment. The MSNC 14, one of the strains (Table 2), was able to degrade 43% of phenanthrene after 30 days of incubation, although the degradation of anthracene and dibenzothiophene was not detected. Al-Mailen et al. [51] isolated four strains (Table 2) from a hypersaline coastal area for the Arabian Gulf on a mineral salt medium containing crude oil vapor as unique source of carbon and energy. The four strains were able to biodegraded not only aliphatic hydrocarbons but also aromatic hydrocarbons after three weeks of incubation. In particular, *Halobacterium* and *Haloccocus* could grow in the presence of benzene, toluene, and p-hydroxybenzoic acid, and the two *Haloferax* strains could grow with toluene and phenanthrene, and one of them also with benzene, but both failed to grow on p-hydroxybenzoic acid. This study also revealed that the biodegradation rates increased in proportion to NaCl concentration in the medium, and thus supported the idea that extreme halophilic archaea are suitable biological material to bioremediate oil-polluted hypersaline environments. Bonfá et al. [63] revealed the usefulness of halophilic archaea in the bioremediation of wastewater of petroleum production, which contained high saline concen‐ tration and various aromatic acids and hydrocarbons. Aromatic compounds (p-hydroxyben‐ zoic acid, naphthalene, phenanthrene, and pyrene) could be also degraded by nine halophilic archaea (Table 2) isolated from Çamalti Saltern, Turkey [64]. This study broadens the under‐ standing of metabolism of aromatic compounds, and the activities of catechol 1,2 dioxygenase and protocatechuate 3,4 dioxygenase were identified as the enzymes involved in *ortho* cleavage pathway. *Ortho* cleavage pathway is widely distributed in soil bacteria and fungi, constituting the major pathway for aromatic compounds catabolism in these organisms. Therefore, these

These results suggest the following: *Halobacteriaceae* family, which can degrade aromatic compounds, exists widely, and can be used to degrade aromatic compounds in oil-polluted hypersaline environments [60]. Thus, the bioremediation process using those strains is

benzoic acid by the *Haloarcula sp.* strain D1 [59].

32 Advances in Bioremediation of Wastewater and Polluted Soil

and *Haloferax sp.* D1227, respectively [62].

enzymes may be important to remove aromatic hydrocarbons.



\*Bertrand et al. [53] labeled this strain as EH4 and assigned it to *Halobacterium sp.* based on phenotypic features. Tapilatu et al. [54] reassigned it to *Haloarcula vallismortis* based on 16 rRNA.

**Table 2.** Extremely halophilic archaea able to biodegrade or grow on aromatic compounds

#### **3.2. Halophilic archaea in bioremediation of heavy metal ion**

Some heavy metals such as iron, cobalt, copper, manganese, molybdenum, and zinc are trace elements required at a certain level, and thus are necessary for life. However, they are excessively damaging to organisms. Other heavy metals such as mercury, aluminium, cadmium, gold, lead, and arsenic are toxic and are not beneficial to organisms. Both essential and nonessential metals at high concentration, directly or indirectly compromise DNA, protein, and membrane integrity and function [67, 68].

Frequently, heavy metals are found in saline and hypersaline environments due to the evaporation in such environments and also as a result of industrial activities. Therefore, some halophilic archaea have developed tolerance to heavy metals. Wang et al. [69] reported that the *Halbacterium sp.* NRC-1, which had a plasmid carrying genes of arsenite and antimonite extrusion system, showed high resistance to arsenic. Kaur et al. [70] studied the haloarchaeal strategies of adaptation to high metal concentration, using *Halobacterium sp.* NRC-1 as model organism. Sublethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) were used to investigate the change on transcriptional level using microarray technology. All growths were inhibited at high concentrations of metals, but the susceptibility was different. Consequently, the effective inhibitory concentrations of Zn (II), Fe (II), Co (II), Cu (II), and Mn (II) were 0.05, 7.5-8.5, 0.6, 1.2, and 2 mM, respectively. Some of the adaptation mechanisms detected include previously known mechanisms such as efflux of metal ions by P1 ATPases, downregulation of Mn(II) uptake, ion scavenging, protein turnover increase, and minimizing ROS production depletion. Novel discoveries include: control of transcriptional regulation by a TRASH domain, ability of ZntA to confer resistance to several metals, a global control mechanism mediated by GTFs and key metalloregulatory proteins, and simulation of Fe deficiency by Mn(II) [70]. Authors developed a systems-level model to provide an integrated perspective of responses to these metals.

Srivastava et al. [71] have reported the intracellular synthesis of silver nanoparticles by the haloarchaeal isolated *Halococcus salifodinae* BK3 when the cells were grown in the medium containing silver nitrate. They also described the intracellular synthesis of selenium nanopar‐ ticles (SeNPs) by the haloarchaeon *Halococcus salifodinae* BK18, when the cells were grown in the presence of sodium selenite. Also, cadmium tolerance has been reported in haloarcheal strains from solar salterns of Ribandar and Siridao in India [73].

Biosorption of metals by the organisms at the surface or by the exopolysaccharides (EPS) secreted to form the biofilms enables organisms to tolerate metals [74]. The adsorption of heavy metal by EPS has been attributed to interaction between metal cations and nega‐ tive charges of acidic functional groups of EPS [75]. Kawakami et al. [76] found that *Halobacterium salinarum* CCM 2090 has a Ca(II)-dependent aggregation system. Calcium ion is adsorbed on the surface of the cells and induces ionic cross-bridging between the EPS, resulting in aggregation of the haloarchaeal cells. Mn2+, Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ could replace Ca2+. However, Mg2+, Sr2+, Mo2+, Cd2+, Sn2+, Hg2+, and Pb2+ induced no flocculation of cells of this halophilic archaeon. In addition, Popescu and Dumitru [75] reported the two *Haloferax* strains had the capacity to reduce the concentration of Pb, Cr, Zn, and Ni ions from media with high salinity, by biosorption process. Knowledge regarding molecular mechanisms underlying resistance to metal is cursory. Therefore, more precise understand‐ ing of the mechanism is necessary to facilitate the use of haloarchaea for bioremediation of metal-polluted hypersaline environments [74].

#### **3.3. Halophilic archaea in bioremediation of wastewater**

**MICROORGANISM AROMATIC COMPOUNDS REFERENCE**

[64]

[64] [56]

[64] [56]

Naphthalene Pyrene Phenanthrene

Naphthalene Pyrene Phenanthrene

Naphthalene Pyrene Phenanthrene

**Table 2.** Extremely halophilic archaea able to biodegrade or grow on aromatic compounds

**3.2. Halophilic archaea in bioremediation of heavy metal ion**

*Haloferax mucosum* Phenanthrene [56] *Haloferax lucentense* Phenanthrene [56] *Haloferax sulfurifontis* Phenanthrene [56]

\*Bertrand et al. [53] labeled this strain as EH4 and assigned it to *Halobacterium sp.* based on phenotypic features. Tapilatu

Some heavy metals such as iron, cobalt, copper, manganese, molybdenum, and zinc are trace elements required at a certain level, and thus are necessary for life. However, they are excessively damaging to organisms. Other heavy metals such as mercury, aluminium, cadmium, gold, lead, and arsenic are toxic and are not beneficial to organisms. Both essential and nonessential metals at high concentration, directly or indirectly compromise DNA,

Frequently, heavy metals are found in saline and hypersaline environments due to the evaporation in such environments and also as a result of industrial activities. Therefore, some halophilic archaea have developed tolerance to heavy metals. Wang et al. [69] reported that the *Halbacterium sp.* NRC-1, which had a plasmid carrying genes of arsenite and antimonite extrusion system, showed high resistance to arsenic. Kaur et al. [70] studied the haloarchaeal strategies of adaptation to high metal concentration, using *Halobacterium sp.* NRC-1 as model organism. Sublethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) were used to investigate the change on transcriptional level using microarray technology. All growths were inhibited at high concentrations of metals, but the susceptibility was different. Consequently, the effective inhibitory concentrations of Zn (II), Fe (II), Co (II), Cu (II), and Mn (II) were 0.05, 7.5-8.5, 0.6, 1.2, and 2 mM, respectively. Some of the adaptation mechanisms detected include previously known mechanisms such as efflux of metal ions by P1 ATPases, downregulation

*Haloarcula hispanica* p-Hydroxybenzoic acid

34 Advances in Bioremediation of Wastewater and Polluted Soil

*Halobacterium salinarum* p-Hydroxybenzoic acid

*Halobacterium piscisalsi* p-Hydroxybenzoic acid

et al. [54] reassigned it to *Haloarcula vallismortis* based on 16 rRNA.

protein, and membrane integrity and function [67, 68].

Wastewater treatments (WWT), such as breakdown of sewage influent, are generally per‐ formed by microorganisms. These microorganisms are able to live in the sludge of treatment plants and holding tanks. They obtain nutrients by degrading the solids in WWT to various compounds. Some wastewater treatment systems are efficient and desirable from an econom‐ ical point of view. However, other systems are not efficient because of the undesirable effects the system itself promote on the microorganisms. Therefore, the establishment of optimal conditions (such as nutrients, pH, temperature, and oxygen availability) for comfortable growth is most important in order to treat WWT effectively.

Modern biological treatment of wastewater involves not only C removal, but also elimination of other nutrients such as P and N. Combined and sequential actions are required for such treatment successively by several groups of microorganisms, such as heterotrophic bacteria, phosphate-accumulating organisms (PAO), or microorganisms able to perform nitrification, denitrification, or anammox [77]. However, it is difficult to design this kind of treatment process, because the system becomes extremely complex to exhibit a satisfactory performance and it requires expensive costs from economical point of view. Figure 1 shows the nitrogen transformation pathways by archaea. Archaea, which can degrade ammonia, are now one of the main candidates for wastewater treatment.

**Figure 1.** Archaeal nitrogen transformations in wastewater treatment environment. The enzymes involved in denitrifi‐ cation process are shown in the figure: NAR, nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase; NOS, nitrous oxide reductase.

Although there are natural microorganisms used in wastewater treatment, the bioremediation process requires further addition of various types of microorganisms known as bioremedia‐ tors. Since there is an organism available to treat any organic molecule (the microorganism does this by extracting the energy from the molecule bonds), it is a very effective treatment. However, it is essential to distinguish the types of microorganisms present in wastewater as well as the pollutants to be removed and where they are to be located in the wastewater process. When these details are known, it is quite easy to select the best microorganisms to be used along with their best locations in the wastewater treatment processing plant.

Figure 2 shows the nitrogen sources produced by artificial activities and their metabolic cycles. The wastewater containing excess nitrogen compounds is constantly discharged from houses and factories, and overall nitrogen species in wastewater and soil are increased. Due to the accumulation of this nitrogen species (NH4 + , NO2 - , and NO3 - , etc.), in groundwater or tap water, the wastewater should be removed or reused [81, 82]. Conventionally, biological nitrogen removal is achieved by nitrification followed by a denitrification process: firstly, aerobic nitrification of NH4 + to NO2 - or NO3 - with O2 as the electron acceptor; secondly, anoxic denitrification of NO2 - or NO3 - to gaseous N2 using organic matter as carbon and energy source [82].

and it requires expensive costs from economical point of view. Figure 1 shows the nitrogen transformation pathways by archaea. Archaea, which can degrade ammonia, are now one of

**Figure 1.** Archaeal nitrogen transformations in wastewater treatment environment. The enzymes involved in denitrifi‐ cation process are shown in the figure: NAR, nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase;

Although there are natural microorganisms used in wastewater treatment, the bioremediation process requires further addition of various types of microorganisms known as bioremedia‐ tors. Since there is an organism available to treat any organic molecule (the microorganism does this by extracting the energy from the molecule bonds), it is a very effective treatment. However, it is essential to distinguish the types of microorganisms present in wastewater as well as the pollutants to be removed and where they are to be located in the wastewater process. When these details are known, it is quite easy to select the best microorganisms to be used

Figure 2 shows the nitrogen sources produced by artificial activities and their metabolic cycles. The wastewater containing excess nitrogen compounds is constantly discharged from houses and factories, and overall nitrogen species in wastewater and soil are increased. Due

or tap water, the wastewater should be removed or reused [81, 82]. Conventionally,

+ , NO2 -

, and NO3


, etc.), in groundwater

along with their best locations in the wastewater treatment processing plant.

to the accumulation of this nitrogen species (NH4

the main candidates for wastewater treatment.

36 Advances in Bioremediation of Wastewater and Polluted Soil

NOS, nitrous oxide reductase.

**Figure 2.** N-cycle scheme. The various sources of pollution of nitrogen cycle species are represented.

Figure 3 shows the typical processes in wastewater plant. Treatments were performed by following three processes:


**c.** Tertiary Treatment: this treatment removes the nitrogen (mainly through denitrification processing) and phosphates (usually by chemical precipitation from the effluent).

Other compounds such as minerals or metals (such as iron, sulfur, manganese), and runoff pollutants such as fertilizers, hydrocarbons, and tar can also be removed. Therefore, extrem‐ ophilic microorganisms able to deal with these compounds have become of great interest in designing new strategies to treat wastewater.

**Figure 3.** Primary, secondary (the tank contains the supernatant followed by primary treatment), and tertiary treat‐ ments used in wastewater treatment plants.

Activated sludge process is currently the most used for the treatment of both domestic and industrial wastewater. The bioreactor has two chambers: one in which the wastewater is agitated and maintained at constant oxygen, where microorganisms degrade the contami‐ nants; second, where the biologically treated water settles to form a sludge that will be removed. Due to the characteristics of the process, the decantation system limits the efficiency of the treatment as a result of problems such as the production of foam, floatability of the sludge, or the large size required for the installation of the wastewater treatment plants [78].

**c.** Tertiary Treatment: this treatment removes the nitrogen (mainly through denitrification processing) and phosphates (usually by chemical precipitation from the effluent).

Other compounds such as minerals or metals (such as iron, sulfur, manganese), and runoff pollutants such as fertilizers, hydrocarbons, and tar can also be removed. Therefore, extrem‐ ophilic microorganisms able to deal with these compounds have become of great interest in

**Figure 3.** Primary, secondary (the tank contains the supernatant followed by primary treatment), and tertiary treat‐

designing new strategies to treat wastewater.

38 Advances in Bioremediation of Wastewater and Polluted Soil

ments used in wastewater treatment plants.

Alternative treatment technologies are thus of great interest when searching for more efficient strategies regarding nutrient removal and the generation of effluents with enough quality for direct reclamation. One new treatment is based on membrane bioreactors (MBRs) (Figure 4): the MBR system consists of a cylindrical bioreactor equipped with two ultrafiltration mem‐ brane units submerged inside the aerated bioreactor, and the extraction of the effluent water takes place by mechanical suction. The membranes are continuously aerated, maintain solids in suspension, and supply oxygen to the process [78].

Submerged biofilters (SBs) (Figure 4) are another reduced-size and low-cost alternative of proven efficiencies for the design of WWTPs, which are of simple control and mainte‐ nance, and minimize undesirable odors and noise in the vicinity of the installations. SBs consist of two methacrylate cylindrical columns each, packed with clayey schists biofilm support [79, 80]. The columns are connected with a valve that allows a separated clean‐ ing of the biofilters. They operate downflow (denitrifying column, anoxic) and upflow (nitrifying column, aerated) [78].

Another recent process for nitrogen removal has gained importance in the last years: the anaerobic ammonium oxidation (anammox). This reaction is based on energy conversion from anaerobic ammonium oxidation using nitrite as the electron acceptor. It was discovered in 1995 in a pilot plant treating wastewater at Gist-Brocades, Delft, in The Netherlands [7, 83]. This process offers a novel, energy-saving and cost-effective biological nitrogen removal technique.

In the past years, partial nitrification, anammox and denitrification simultaneously in a single reactor (SNAD technology), was developed for the complete removal of nitrogen sources. Under oxygen limitation, ammonium is oxidized to nitrite by aerobic ammonium oxidation; the nitrite in the reactor can be used by anammox microorganisms with ammonium, and finally to dinitrogen gas with small amounts of nitrate produced. Afterward, COD as electron donor can deoxidize nitrate to dinitrogen gas through denitrifying process for the complete nitrogen removal performance. The interaction of aerobic nitrifying, anaerobic ammonium oxidizing, and anaerobic denitrifying microorganisms under oxygen limitation has the potential to make an almost complete conversion of ammonium and organic carbon to dinitrogen gas and carbon dioxide [84].

Although the importance of eukaryotic and bacteria organisms in aerated activated sludge has long been recognized, the role played by archaea in aerobic and anaerobic WWPTs has not been mentioned. However, recent researches suggest that growth and activity of archaea were significant in the treatment of activated sludge and wastewater [85-87]. The roles of methano‐ genic archaea within a broad range of activated sludge, submerged biofilters, and membrane

**Figure 4.** Schematic diagram of the two new types of WWTPs. (A) Submerged membrane bioreactor (MBR). (B) Sub‐ merged biofilter (SB).

bioreactors have been studied in recent research [78, 88-89]. Under oxic conditions, no methanogenesis was detected; however, once oxygen is depleted, methane production ensued. The results suggest that methanogenic archaea can be activated under anoxic conditions [88].

The composition of the wastewater is a key determinant of archaeal community composition in WWPTs [88, 90-91]: the microbial population in industrial wastewater treatments, rich in ammonia, phenol, and with high salinity, are closely related to *Methanobrevibacter smithii*, the predominant methanogen in human intestines [78]. The manufacturing of chemical com‐ pounds (pesticides, herbicides, explosives, etc.) usually generates effluents containing complex mixtures of salts and nitrate or nitrite. Also, the increase of salinity in soils and waters in the last few decades has given advantage to some species like *Hfx. mediterranei.* For example, *Hfx. mediterranei* is resistant to very high nitrate (up to 2 M) and nitrite (up to 50 mM) concen‐ trations, which are the highest described from a prokaryotic microorganism [92]. Therefore, it could be useful for bioremediation applications in sewage plants where high salts, nitrate, and nitrite concentrations are detected in wastewaters and brines. In a recent study [93], this haloarchaea was able to eliminate 60% of the nitrate and 75% of nitrite initially present in the brines (initial concentration was 40 mM nitrite). Moreover, it has also been described that nitrate reductase involved in denitrification reduces efficiently other oxyanions such us bromate and (per) chlorate [94]. These results suggest that *Hfx. mediterranei*, and in general, halophilic archaea, are able to carry out denitrification, thus providing excellent models to explore large-scale bioremediation processes to remove nitrogen compounds from brines and salty water.
