Heavy metals are among the most persistent and toxic pollutants in the environment [1]. Even in small concentrations, they can threat human health as well as the environment because they are non-biodegradable. There is no widely agreed criterion for definition of a heavy metal. Depending on the context, this term can acquire different meanings: for example, in metallurgy a heavy metal may be defined by its density, in physics by its atomic number, and in chemistry by its chemical behavior [2]. The International Union of Pure and Applied Chemistry (IUPAC) does not consider these definitions correct; in this review, according to IUPAC, every “heavy” metal has the following characteristics: density exceeding 5.0 g/cm³; general behavior as cations; low solubility of their hydrates; aptitude to form complexes and affinity towards the sulfides.

In 2010, the World Health Organization (WHO) estimated that more than 25% of total diseases were linked to environmental factors including exposure to toxic chemicals [3]. For example, lead, [Pb(II)], one of the most common heavy metals, is thought to be responsible for 3% of cerebrovascular disease worldwide [4]; while cadmium, [Cd(II)], has been classified as carcinogen by the International Agency for Research on Cancer (IARC) on the basis of several evidence in both humans and experimental animals [5, 6]. Furthermore, hazards associated with exposure to other metal ions like chromium [Cr(II)], mercury [Hg(I)], and arsenic [As(III) and As(V)], have been well established in the literature [7-12]. The risk related to heavy metal exposure depends on the concentration and time [13].

Table 1 reports the concentration limits of the most common heavy metals in drinkable water, suggested by the World Health Organization (WHO) and the Environmental Protection Agency (EPA). It also shows the possible sources of these contaminants in drinking water and the potential health effects from long-term exposure [12, 14].

On the other hand, heavy metals naturally occur in the Earth’s crust. They are present in soils, rocks, sediments, air and waters and can be used and modified by local microbial communities, which are actively involved in metal geochemical cycles, affecting their speciation and mobility. Many metals are essential for life because they are actively involved in almost all aspects of metabolism: as examples, iron and copper are involved in the electron transport, manganese and zinc influence enzymatic regulations. However, their excess can disrupt natural biochemical processes and cause toxicity. For these reasons, all the microorganisms have evolved resistance systems to get rid of the cell of toxic metals as well as molecular mechanisms to maintain metal homeostasis. These systems frequently rely on a balance between uptake and efflux processes [15]. Because of microbial adaptation, microorganisms can also contribute to increase toxicity levels [16, 17]. For example, several studies in Bangladesh have demonstrated that microbial processes enhance the arsenic contamination in near- and sub-surface aquifers, because arsenate-respiring bacteria can liberate As(III) from sediments, adsorptive sites of aluminum oxides or ferrihydrite, or from minerals, such as scorodite [18].

Metal biotransformation impacts human health through the food chain: examples include the oxidation of Hg(0) to Hg(II), and the subsequent methylation to methylmercury compounds, which can be accumulated by fish and marine mammals in the aquatic environment [19].

Despite their relevant toxicity, in a report of the European Commission (named “Critical-Metals in the Path towards the Decarbonisation of the EU Energy Sector”), several heavy metals such as cadmium, chromium and lead are included into the classification of critical raw materials. According to the sustainable low-carbon economic policy of EU, these metals are expected to become a bottleneck in a near future to the supply- chain of various low- carbon energy technologies [20]. Therefore, it is very important to detect and recover these heavy metals to achieve both environmental safeguard and sustainable economic strategies. Common sources of heavy metals in this context include mining and industrial wastes, vehicle emissions, lead-acid batteries, fertilizers, paints, treated timber, aging water supply infrastructures, and microplastics floating in the world's oceans [21, 22].

Microorganisms able to tolerate high levels of heavy metal ions have evolved in ore deposits, hydrothermal vents, geothermal sites, as well as in different polluted sites [23]. Metal tolerance of thermophilic Bacteria/Archaea is due to several mechanisms, many also found in mesophilic counterparts, such as: extracellular barrier, metal ion transport into and outside the cell, the utilization of toxic metal ions in metabolism or the presence of metal resistance genes with different genomic localization (chromosome, plasmid or transposon) [24].

To date, at least four main mechanisms of heavy metal resistance, schematized in Fig. (1), are described which can be even found in the same microorganism [24-31]:

Fig. (1). Generalized illustration of the genetic mechanism of resistance to toxic metals by microorganisms: 1) Extracellular barrier, a selectively permeable system; 2) Efflux of metal ions; 3) Enzymatic reduction of metal ions. 4) Intracellular sequestration by small molecule complexing agents or metal-chelating proteins.

• Extracellular barrier;
• Active transport of metal ions (efflux);
• Enzymatic reduction of metal ions;
• Intracellular sequestration.

The cell wall or plasma membrane can prevent metal ions from entering the cell. Bacteria belonging to different taxonomical groups can adsorb metal ions by ionisable groups of the cell wall (carboxyl, amino, phosphate and hydroxyl groups) [32]. However, many metal ions enter the cell via the systems responsible for the uptake of essential elements: for example, Cr(II) is transported inside the cell via sulphate transport system [33], whereas Cd(II), Zn(II), Co(II), Ni(II) and Mn(II), enter the cells using systems of magnesium transport [34]. Moreover, As(V) is taken into cells by phosphate transport systems and As(III) has been shown to be taken up by glucose permeases [35].

Both in Gram-positive and Gram-negative bacteria the arsenic resistance system is composed by operons of 3-5 genes carried on plasmids or chromosomes; the two most common contain either five genes (arsRDABC), as in the plasmid R773 of the Escherichia coli, or three genes (arsRBC), as in the plasmid pI258 of Staphylococcus aureus [36, 37]. The arsR gene encodes a trans-acting repressor of the ArsR/SmtB family involved in transcriptional regulation [30], arsB encodes an As(III) efflux transporter [38], and arsC encodes a cytoplasmic arsenate reductase that converts As(V) to As(III), which is extruded outside the cell [39]. Where present, ArsD is a metallochaperone that transfers trivalent metalloids to ArsA, the arsenite-stimulated efflux ATPase [31].

A detailed understanding of the molecular mechanisms responsible for resistance to toxic metals in metal-tolerant microorganisms is also crucial for a potential use in the environmental monitoring of metal contamination and to set up bioremediation processes, the most promising being biosorption and removal as insoluble complexes Fig. (2).

Fig. (2). Schematic representation of potential applications in biotechnology of metal-tolerant thermophiles.

The traditional approach for monitoring the environmental pollution is based on chemical or physical analysis and allows highly accurate and sensitive determination of the exact composition of any sample. These analyses require specialized and expensive instrumentations, as the ICP-MS (Inductively Coupled Plasma - Mass Spectrometry) which, to date, is the most adopted technique for detecting heavy metals [63].

The need for accurate, not expensive, on-site and real-time measurements has led to the development of sensors based on biomolecules and nanomaterials [63-66]. Biosensors are analytical devices which integrate a biological recognition element with a physical transducer to generate a measurable signal proportional to the concentration of the analyte [67-69]. The use of biological molecules is a considerable advantage in the sensor field, because of their high specificity: these sensors are based on the specific interaction between enzymes and their substrates, antibodies and antigens, target molecules to their receptors, or the high affinity of nucleic acid strands to their complementary sequences [67]. Nevertheless, some biomolecules can be too labile for the exploitation on the marketplace. In this context, the biomolecules of the thermophiles are more stable at high temperatures than the mesophilic counterparts. In fact they have already been exploited in biotechnology, as demonstrated by the development of the polymerase chain reaction (PCR) and the use of thermozymes in many industrial applications [70-73].

The recent developments in nanotechnology have also opened new horizons for biosensing: nanomaterials are attractive because of their unique electrical, chemical and physical properties (i.e. size, composition, conductivity, magnetism, mechanical strength, and light-absorbing and emitting). The most studied of them, carbon nanotubes (CNTs), graphene, metal nanoparticles (MNPs), and quantum dots (QDs), have been especially targeted for developing novel biosensors [64, 73-76]. An example of a successful match between thermophilic biomolecules and nanomaterial for the development of a heavy metal biosensor is reported by Politi et al. [77, 78]. In this work, TtArsC, the arsenate reductase from T. thermophilus HB27 [54] was conjugated to polyethylene glycol-stabilized gold nanospheres. The new nanobiosensor revealed high sensitivities and limits of detection equal to 10 ± 3 M^-12 and 7.7 ± 0.3 M^-12 for As(III) and As(V), respectively [77, 78].

A detailed understanding of the molecular mechanisms responsible for resistance to toxic metals is crucial to develop whole cell biosensors for the detection of chemicals in the environment so that not only thermophilic biomolecules, but also thermophilic microorganisms can constitute heavy metal biosensors. Many whole-cell biosensors for metal ions detection have been already described in the literature based on the realization of reporter systems containing regulatory cis-acting sequences interacting with the cognate transcriptional metal sensor repressor; however, to date, there are few reports on thermophilic whole cell biosensors [79, 80].

In the work by Poli et al. [57], Anoxybacillus amylolyticus, an acidothermophilic bacterium isolated from geothermal soil samples in Antarctica, was observed to be resistant to metals like Ni(II), Zn(II), Co(II), Hg(II), Mn(II), Cr(VI), Cu(II) and Fe(III). A decrease in α-amylase activity, correlated with a decrease in α-amylase production, was observed in the presence of heavy metals: it is speculated to use it as a toxicological indicator of heavy metals in a potential microbial bioassay employing whole cells [57].

Biomining and bioremediation represent a new branch of biotechnology, named biometallurgy, addressed at heavy metal recovery including the processes that involve interactions between microorganisms and metals or metal-bearing minerals [81]. Biomining refers to the exploitation of microorganisms to extract and recover metals from ores and waste concentrates (the term is often used synonymously with bioleaching when the metals are solubilized during the process); on the other hand, bioremediation focuses on the transformation of a toxic substance into a harmless or less toxic one from contaminated sites [82, 83]. In this contest, among the heavy metals resistance systems developed by microorganisms, and in particular by thermophiles, biosorption and bioaccumulation are emerging as promising low-cost methodologies for bioremediation [84, 85]. Biosorption and bioaccumulation are two processes that consist into the ability of microorganisms to accumulate heavy metals from wastewater through metabolic pathways or physical-chemical uptake; but while biosorption is a passive process depending on the composition of the cellular surface and following a kinetic equilibrium, bioaccumulation is an energy driven process and requires an active metabolism [59].

Several examples of biosorption and bioaccumulation are provided by microorganisms belonging to Geobacillus sp., which are highly tolerant to Cd(II), Cu(II) and Zn(II) [86-88]. Özdemir, S. et al, indicated a G. toebii subsp. decanicus as an efficient viable biosorbent for heavy metals. This study clearly shows that thermophiles can be used for removal and recovery of heavy metals from industrial wastewater [89].

The interest into the potential applications of heavy metal resistant thermophiles has led to the development of tools and assays for screening them on lab scale [61, 90-92], with the final goal to design and set up bacterial bioprocesses on the industrial scale [93-95]. Table 2 summarizes some examples of bioprocesses employing heavy metals performed by thermophiles.

Metal-tolerant thermophiles exhibit metabolic and physiological features that distinguish them from other major life groups due to their adaptation to extreme environments.

Most of the knowledge regarding mechanisms of adaptation/resistance to toxic metals has been discovered using traditional microbiological/biochemical techniques and thanks to the use of genomics, transcriptomics, proteomics as well as to the recent development of genetic tools for many of these organisms. With the advent of next-generation sequencing technologies, comparative genomics and metagenomics projects, it appears that even novel metabolic features can be discovered, further expanding our understanding of environmental microbiology. Such an integrated view opens to new opportunities for biotechnological applications in commercially relevant processes such as the monitoring of metal concentrations in the environment, the recovery of precious and strategic metals and the setup of microbial-based remediation strategies.