**2. General characteristics of archaeal species and their potential availabilities to support bioremediation strategies**

The word "archaea" means "ancient things" (from Greek) and it refers to a group of prokary‐ otic single-celled microorganisms characterized for the extreme conditions they need to be alive. Archaea, which are single-celled prokaryotic microorganisms, were first classified as a separate group of prokaryotes in 1977 by Woese and Fox [10]. Extreme conditions are necessary for archaea to live [10] and extremophiles such as methanogens, thermoacidophiles, halo‐ philes, or alkalophilic microorganisms are included in the group.

Haloarchaea (salt-loving organisms) can grow in media with high salt concentration in a range of 12% to 30% salt (2-5 M NaCl). The cellular machinery of haloarchaea can work even in such high concentration of salt, because it accumulates potassium ion to counteract high concen‐ tration of sodium ion. Whereas biodegradation and bioremediation by non-extremophilic microorganisms have been extensively study, the use of extremophilic microorganisms is less studied and particularly haloarchaea.

Enzymes and metabolic pathways specific in archaea were suggested by the comparison between genome sequences of archaea and others [11]. The metabolic pathways of carbohy‐ drates, carbon and nitrogen assimilation or fixation, and sulfur metabolism were involved in the specific pathways, which are different from classical pathways existing in bacteria and eukarya. The enzymes of archaea may be used for the bioremediation.

## **2.1. Carbohydrate metabolism**

technologies and the hazards of conventional soil cleanup methods have spurred investiga‐ tions into in situ bioremediation, which uses microorganisms to destroy or immobilize

In developing countries, 90% of untreated wastewater goes into the rivers so the access to safe drinking water is limited. The increasing water demand not only affects surface freshwater like rivers and lakes, but it also degrades groundwater resources [5]. The eutrophication and the presence of excess nitrogen in the environment have caused serious alterations of the natural nutrient cycle between the living world and the soil, water, and atmosphere [5]. The intensification of agricultural production and continuous industrial development have contributed to an increase in the nitrate content in drinking water. This is particularly evident in rural areas, where in private wells the concentration of nitrate nitrogen is often over twenty times above the permissible level. Therefore, it is now necessary to develop a technology which

The engineering of bioremediation processes relies on information about the site and about candidate microorganisms. Process analysis usually begins with fixed waste characteristics but with options for microbial cultures, reactor types, waste pretreatment, and process operating conditions. Laboratory measurements are necessary to explore these options and to design an efficient process. These tests examine degradation rates as functions of critical operating parameters such as pH, oxygen and nutrient concentrations, microbial composition, soil particle size, temperature, and redox potential, shaping the design of a bench-scale process. Mass transfer effects such as agitation and aeration are also explored, although at a small scale. These tests constitute the basis for scale-up to the field scale and for the implementation of process control [8]. A main objective of biological remediation design is to remove the limiting factors in the growth of bacteria [9]. The main objective of site characterization is to identify

effectively reduces nitrate concentration in drinking water [7].

the contaminants, their concentration, and the extent of contamination.

**availabilities to support bioremediation strategies**

philes, or alkalophilic microorganisms are included in the group.

studied and particularly haloarchaea.

**2. General characteristics of archaeal species and their potential**

The word "archaea" means "ancient things" (from Greek) and it refers to a group of prokary‐ otic single-celled microorganisms characterized for the extreme conditions they need to be alive. Archaea, which are single-celled prokaryotic microorganisms, were first classified as a separate group of prokaryotes in 1977 by Woese and Fox [10]. Extreme conditions are necessary for archaea to live [10] and extremophiles such as methanogens, thermoacidophiles, halo‐

Haloarchaea (salt-loving organisms) can grow in media with high salt concentration in a range of 12% to 30% salt (2-5 M NaCl). The cellular machinery of haloarchaea can work even in such high concentration of salt, because it accumulates potassium ion to counteract high concen‐ tration of sodium ion. Whereas biodegradation and bioremediation by non-extremophilic microorganisms have been extensively study, the use of extremophilic microorganisms is less

contaminants in place.

26 Advances in Bioremediation of Wastewater and Polluted Soil

In archaea, the modified version of the Embden-Meyerhoff-Parnas and Entner-Doudoroff pathways, and pentose degradation pathway have been described [12]. The Embden-Meyerh‐ off-Parnas is an optimized pathway of glycolysis for the conversion/oxidation of glucose to two molecules of pyruvate yielding ATP and intermediates for other metabolic pathways. The main modifications in this pathway include the presence of the enzymes as ADP-dependent glucokinase and phosphofructokinase, phosphoenolpyruvate synthase, pyruvate:phosphate dikinase or nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. This modified pathway is present, for example, in members of *Thermococcales* [13], *Archaeoglobales* [14], and *Desulfurococcales* [15]. The main difference of the classical Entner-Doudoroff pathway is that this pathway is divided into two branches: semi-phosphorylated or nonphosphorylated. The highest enzymes of both branches are phosphoenolpyruvate synthase, pyruvate:phosphate dikinase and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, as in the Embden-Meyerhoof-Parnas pathway. The semi-phosphorylated pathway is found in hal‐ oarchaea [16], and the nonphosphorylated in the genera *Thermoplasma* [17], *Sulfolobus*[18], and *Thermoproteus* [19].

Pentoses are ubiquitous in nature as part of nucleic acids. The degradation of pentoses has been described in some haloarchaea [16, 20] and species for the genera *Sulfolobus* [21]. This pathway in archaea is similar to that identified in bacteria.

#### **2.2. Carbon fixation/assimilation**

Regarding the assimilation of carbon, different metabolic pathways have been described in archaea. Acetyl-CoA assimilation was performed by ethylmalonyl-CoA pathway and the glyoxylate cycle. In addition to this glyoxylate cycle, several haloarchaea can assimilate acetate [12]. Recently, Khomyakova et al. [22] have proposed a new acetyl-CoA assimilation pathway in *Haloarcula marismortui*, the methyl aspartate cycle.

Metabolic studies of autotrophic archaea have led to the discovery of several different CO2 fixation pathways such as the reductive tricarboxylic acid cycle, the Calvin-Benson-Bassham cycle, the 3-hydroxypropionate cycle, and the reductive acetyl-CoA pathway. In anaerobic or microaerobic *Thermoproteales* and *Desulfurococcales,* the dicarboxilate-4-hydroxybutirate cycle is present [23,24]. The oxygen sensibility of some of the enzymes of this cycle restricts its use to this kind of microorganism. The hydroxypropionate–hydroxybutirate cycle was identified in members of the order *Sulfolobales* [25].

#### **2.3. Nitrogen 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 candidate microorganism for bioremediation approaches [26-28]..

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 [39] and some thermophiles [40].

#### **2.4. Sulfur compounds metabolism**

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