Iron bioleaching and polymers accumulation by an extreme acidophilic bacterium.

In many European regions, both local metallic and non-metallic raw materials are poorly exploited due to their low quality and the lack of technologies to increase their economic value. In this context, the development of low cost and eco-friendly approaches, such as bioleaching of metal impurities, is crucial. The acidophilic strain Acidiphilium sp. SJH reduces Fe(III) to Fe(II) by coupling the oxidation of an organic substrate to the reduction of Fe(III) and can therefore be applied in the bioleaching of iron impurities from non-metallic raw materials. In this work, the physiology of Acidiphilium sp. SJH and the reduction of iron impurities from quartz sand and its derivatives have been studied during growth on media supplemented with various carbon sources and under different oxygenation conditions, highlighting that cell physiology and iron reduction are tightly coupled. Although the organism is known to be aerobic, maximum bioleaching performance was obtained by cultures cultivated until the exponential phase of growth under oxygen limitation. Among carbon sources, glucose has been shown to support faster biomass growth, while galactose allowed highest bioleaching. Moreover, Acidiphilium sp. SJH cells can synthesise and accumulate Poly-ß-hydroxybutyrate (PHB) during the process, a polymer with relevant application in biotechnology. In summary, this work gives an insight into the physiology of Acidiphilium sp. SJH, able to use different carbon sources and to synthesise a technologically relevant polymer (PHB), while removing metals from sand without the need to introduce modifications in the process set up.

Enhancing column bioleaching of chalcocite by isolated iron metabolism partners Leptospirillum ferriphilum/Acidiphilium sp. coupling with systematically utilizing cellulosic waste.

The iron metabolism partners Leptospirillum ferriphilum and Acidiphilium sp. were screened from industrial bioheap site. An integrated multi-stage strategy was proposed to improve chalcolite column bioleaching coupling with synergistical utilization of cellulosic waste such as acid hydrolysate of aquatic plants. L. ferriphilum was used to accelerate the initial iron metabolism, and Acidithiobacillus caldus maintained a lower pH in the middle stage, while Acidiphilium sp. greatly inhibited jarosite passivation in the later stage. Meanwhile, L. ferriphilum (38.3 %) and Acidiphilium sp. (37.0 %) dominated the middle stage, while the abundance of Acidiphilium sp. reached 63.5 % in the later stage. The ferrous, sulfate ion and biomass were improved and the transcriptional levels of some biofilm and morphology related genes were significantly up-regulated. The final Cu2+ concentration reached 325.5 mg·L-1, improved by 43.8 %. Moreover, Canonical Correlation Analysis (CCA) analysis between bioleaching performance, iron/sulfur metabolism and community verified the important role of iron metabolism partners.

Microbial biominers: Sequential bioleaching and biouptake of metals from electronic scraps.

Electronic scraps (e-scraps) represent an attractive raw material to mine demanded metals, as well as rare earth elements (REEs). A sequential microbial-mediated process developed in two steps was examined to recover multiple elements. First, we made use of an acidophilic bacteria consortium, mainly composed of Acidiphilium multivorum and Leptospidillum ferriphilum, isolated from acid mine drainages. The consortium was inoculated in a dissolution of e-scraps powder and cultured for 15 days. Forty-five elements were analyzed in the liquid phase over time, including silver, gold, and 15 REEs. The bioleaching efficiencies of the consortium were >99% for Cu, Co, Al, and Zn, 53% for Cd, and around 10% for Cr and Li on Day 7. The second step consisted of a microalgae-mediated uptake from e-scraps leachate. The strains used were two acidophilic extremotolerant microalgae, Euglena sp. (EugVP) and Chlamydomonas sp. (ChlSG) strains, isolated from the same extreme environment. Up to 7.3, 4.1, 1.3, and 0.7 µg by wet biomass (WB) of Zn, Al, Cu, and Mn, respectively, were uptaken by ChlSG biomass in 12 days, presenting higher efficiency than EugVP. Concerning REEs, ChlSG biouptake 14.9, 20.3, 13.7, 8.3 ng of Gd, Pr, Ce, La per WB. Meanwhile, EugVP captured 1.1, 1.5, 1.4, and 7.5, respectively. This paper shows the potential of a microbial sequential process to revalorize e-scraps and recover metals and REEs, harnessing extremotolerant microorganisms.

In silico genome analysis of an acid mine drainage species, Acidiphilium multivorum, for potential commercial acetic acid production and biomining.

The genome of Acidiphilium multivorum strain AIU 301, acidophilic, aerobic Gram-negative bacteria, was investigated for potential metabolic pathways associated with organic acid production and metal uptake. The genome was compared to other acidic mine drainage isolates, Acidiphilium cryptum JF-5 and Acidithiobacillus ferrooxidans ATCC 23270, as well as Acetobacter pasteurianus 386B, which ferments cocoa beans. Plasmids between two Acidiphilium spp. were compared, and only two of the sixteen plasmids were identified as potentially similar. Comparisons of the genome size to the number of protein coding sequences indicated that A. multivorum and A. cryptum follow the line of best fit unlike A. pasteurianus 386B, which suggests that it was improperly annotated in the database. Pathways between these four species were analyzed bioinformatically and are discussed here. A. multivorum AIU 301, shares pathways with A. pasteurianus 386B including aldehyde and alcohol dehydrogenase pathways, which are used in the generation of vinegar. Mercury reductase, arsenate reductase and sulfur utilization proteins were identified and discussed at length. The absence of sulfur utilization proteins from A. multivorum AIU 301 suggests that this species uses previously undefined pathways for sulfur acquisition. Bioinformatic examination revealed novel pathways that may benefit commercial fields including acetic acid production and biomining.

The significance of pH in dictating the relative toxicities of chloride and copper to acidophilic bacteria.

The ability of acidophilic bacteria to grow in the presence of elevated concentrations of cationic transition metals, though varying between species, has long been recognized to be far greater than that of most neutrophiles. Conversely, their sensitivity to both inorganic and organic anions, with the notable exception of sulfate, has generally been considered to be far more pronounced. We have compared the tolerance of different species of mineral-oxidizing Acidithiobacillus and Sulfobacillus, and the heterotrophic iron-reducer Acidiphilium cryptum, to copper and chloride when grown on ferrous iron, hydrogen or glucose as electron donors at pH values between 2.0 and 3.0. While tolerance of copper varied greatly between species, these were invariably far greater at pH 2.0 than at pH 3.0, while their tolerance of chloride showed the opposite pattern. The combination of copper and chloride in liquid media appeared to be far more toxic than when these elements were present alone, which was thought to be due to the formation of copper-chloride complexes. The results of this study bring new insights into the understanding of the physiological behaviour of metal-mobilising acidophilic bacteria, and have generic significance for the prospects of bioleaching copper ores and concentrates in saline and brackish waters.

Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling.

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Spectroscopic study on biological mackinawite (FeS) synthesized by ferric reducing bacteria (FRB) and sulfate reducing bacteria (SRB): Implications for in-situ remediation of acid mine drainage.

Mackinawite (FeS), widespread in low temperature aquatic environments, is generally considered to be the first Fe sulfide formed in sedimentary environments which has shown effective immobilization of heavy metals and toxic oxyanions through various sorption reactions. The spectroscopic study researches on mackinawite formed by FRB and SRB and its environmental implication for in-situ remediation of acid mine drainage where contains large amounts of Fe3+ and SO42-. The XRD result of biologically synthetic particles shows that these particles are mainly composed of mackinawite (FeS0.9). The Raman peaks observed at 208, 256, 282, 298cm-1 are attributed to FeS stretching vibrations of mackinawite. The Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) reveals that the diagnostic bands of low intensity for these FeS particles occur at 412-425cm-1 and 607-622cm-1, which are assigned to the stretching vibrations of SS and FeS bonds. The Raman and IR vibrations from organic components both confirm that these particles are biogenic origin. The IR spectra of biologically synthesized mackinawite for different aging times show that the nano-sized particles mackinwate will be completely oxidized within 10h. All these findings have good implications for in-situ remediation of acid mine drainage.

Solid and liquid media for isolating and cultivating acidophilic and acid-tolerant sulfate-reducing bacteria.

Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro The main features of the 'standard' solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors.

Putative bacterial interactions from metagenomic knowledge with an integrative systems ecology approach.

Following the trend of studies that investigate microbial ecosystems using different metagenomic techniques, we propose a new integrative systems ecology approach that aims to decipher functional roles within a consortium through the integration of genomic and metabolic knowledge at genome scale. For the sake of application, using public genomes of five bacterial strains involved in copper bioleaching: Acidiphilium cryptum, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans, we first reconstructed a global metabolic network. Next, using a parsimony assumption, we deciphered sets of genes, called Sets from Genome Segments (SGS), that (1) are close on their respective genomes, (2) take an active part in metabolic pathways and (3) whose associated metabolic reactions are also closely connected within metabolic networks. Overall, this SGS paradigm depicts genomic functional units that emphasize respective roles of bacterial strains to catalyze metabolic pathways and environmental processes. Our analysis suggested that only few functional metabolic genes are horizontally transferred within the consortium and that no single bacterial strain can accomplish by itself the whole copper bioleaching. The use of SGS pinpoints a functional compartmentalization among the investigated species and exhibits putative bacterial interactions necessary for promoting these pathways.

Use of an acidophilic yeast strain to enable the growth of leaching bacteria on solid media.

In this study, a Candida digboiensis strain was isolated from a heap leaching plant in Zambia and used in double-layer agar plate to efficiently isolate and purify leaching bacteria. Unlike Acidiphilium sp., the yeast strain was tetrathionate tolerant and could metabolize a great range of organic compounds including organic acids. These properties allowed the yeast strain to enable and fasten the growth of iron and sulfur oxidizers on double-layer agar plate. The isolates were identified as Acidithiobacillus ferrooxidans FOX1, Leptospirillun ferriphilum BN, and Acidithiobacillus thiooxidans ZMB. These three leaching bacteria were inhibited by organic acids such as acetic and propionic acids; however, their activities were enhanced by Candida digboiensis NB under dissolved organic matter stress.

Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

The effect of energy substrates on PHB accumulation of Acidiphilium cryptum DX1-1.

The effect of glucose and elemental sulfur on the growth and PHB accumulation of Acidiphilium cryptum DX1-1 was investigated. Meanwhile, the differential expressions of 19 genes related with PHB accumulation, sulfur metabolism and carbon fixed in heterotrophy, phytotrophy and mixotrophy were studied by RT-qPCR. The results showed that strain DX1-1 could accumulate PHB with sulfur as the energy substance and atmospheric CO2 as carbon resource. Glucose could improve the growth of strain DX1-1 cultured in medium with sulfur as the energy substance, and almost all the key enzyme-encoding genes related with PHB, sulfur metabolism and carbon fixed were basically up-regulated. PHB polymerase (Arcy_3030), ribulose-bisphosphate carboxylase (Acry_0825), ribulose-phosphate-epimerase (Acry_0022), and cysteine synthase A (Acry_2560) played important role in PHB accumulation, the modified expression of which could influence the PHB yield. With CO2 as carbon resource, the main initial substance of PHB accumulation for strain DX1-1 was acetyl-CoA, instead of acetate with the glucose as the carbon resource. Because of accumulating PHB by fixed atmospheric CO2 while independent of light, A. cryptum DX1-1 may have specifically potential in production of PHB.

Mechanism of cadmium binding on the cell wall of an acidophilic bacterium.

Under certain conditions bacteria can act as a good biosorbent for different toxic heavy metals. However, no study on this aspect has been reported in case of acidophilic, heterotrophic, Gram-negative Acidiphilium strains, which are mostly resistant to several heavy metals. FTIR, SEM, TEM along with sorption experiments using bacterial cells of Acidiphilium symbioticum H8 were conducted to establish the mechanism of Cd(2+) ion sorption. The anionic functional groups present in the cell envelop were the components primarily responsible for the metal-binding capability of the bacterium. Sorption experiments further confirmed that 248.62 mg of cadmium was adsorbed per gram biomass at pH 6.0. The process can better be explained by Langmuir-Freundlich dual isotherm model. Blocking of the functional groups by chemical modification suggested that the binding of cadmium on the biomass occurs through electrostatic reaction and complex formation. Accumulation of cadmium on the cell envelop was supported by fine structure study.

The co-culture of Acidithiobacillus ferrooxidans and Acidiphilium acidophilum enhances the growth, iron oxidation, and CO2 fixation.

Although the synergetic interactions between chemolithoautotroph Acidithiobacillus ferrooxidans and heterotroph Acidiphilium acidophilum have drawn a share of attention, the influence of Aph. acidophilum on growth and metabolic functions of At. ferrooxidans is still unknown on transcriptional level. To assess this influence, a co-culture composed by At. ferrooxidans and Aph. acidophilum was successfully acclimated in this study. Depending on the growth dynamics, At. ferrooxidans in co-culture had 2 days longer exponential phase and 5 times more cell number than that in pure culture. The ferrous iron concentration in culture medium and the expression of iron oxidation-related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Besides, the analysis of CO2 fixation-related genes in At. ferrooxidans indicated that the second copy of RuBisCO-encoding genes cbbLS-2 and the positive regulator-encoding gene cbbR were up-regulated in co-culture system. All of these results verified that Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation-related genes and the second set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2 fixation by At. ferrooxidans.

1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum.

We report the (1)H, (13)C, and (15)N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, isolated from the acidophilic gram-negative facultatively anaerobic metal-reducing alphaproteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c (2) and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on (13)C- and (15)N- labeled substrates.

Mechanism of adsorption of ferric iron by extracellular polymeric substances (EPS) from a bacterium Acidiphilium sp.

The aim of this study was to assess the sorption of Fe(III) by extracellular polymeric substances (EPS) of the Acidiphilium 3.2Sup(5) bacterium, which has promising properties for use in microbial fuel cells (MFC). The EPS of A. 3.2Sup(5) was extracted using EDTA. The sorption isotherms were determined using aliquots of purified EPS. The exosubstances loaded with metal were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction spectroscopy (XRD) and Fourier transform infrared spectroscopy (FTIR). The sorption uptake approaches to 536.1 +/- 26.6 mg Fe(III) (g EPS)(-1) at an initial ferric concentration of 2.0 g l(-1). The sorption of Fe(III) by EPS can be fitted to the Freundlich model. The sorption process produces hydrated iron (III) oxalate [Fe(OH)(C2O4) x 2H2O] by a reversible reaction (log K = 1.06 +/- 0.16), indicating that a shift in the sorption of the cation can be easily achieved. Know the magnitude and form of iron sorption by EPS in MFC can foresee the potential impact on the metabolism of iron-reducing and iron-oxidazing bacteria and, therefore, on the feasibility of the system.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

Real-time PCR analysis of metabolic pathway of PHB in Acidiphilium cryptum DX1-1.

The time, yield and related genes expression of PHB accumulation of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios 1.2, 2.4, 7.5, and 24. The results of time and yield of PHB accumulation show that the initial C/N ratio 2.4 was optimum for strain DX1-1 to accumulate PHB, both higher and lower initial C/N ratios did not favor that process. Based on the genome of Acidiphilium cryptum JF-5, 13 PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expression of the 13 functional genes, in different C/N ratios as cited above, was then studied by Real-time PCR. The results show that all the 13 genes were most upregulated when initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-beta-hydroxybutyrate polymerase and Acry_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which prove that they play the most important role for PHB accumulation and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis shows that the genes related to PHB accumulation are regulated by different promoters and that the motif had weak similarity to the model promoters, suggesting that PHB- metabolism in Acidiphilium cryptum may be mediated by a different mechanism.

Microbial architecture of environmental sulfur processes: a novel syntrophic sulfur-metabolizing consortia.

Microbial oxidation of sulfur-rich mining waste materials drives acid mine drainage (AMD) and affects the global sulfur biogeochemical cycle. The generation of AMD is a complex, dynamic process that proceeds via multiple reaction pathways. The role of natural consortia of microbes in AMD generation, however, has received very little attention despite their widespread occurrence in mining environments. Through a combination of geochemical experimentation and modeling, scanning transmission X-ray microscopy, and fluorescent in situ hybridization, we show a novel interdependent metabolic arrangement of two ubiquitous and abundant AMD bacteria: chemoautotrophic sulfur-oxidizing Acidithiobacillus sp. and heterotrophic Acidiphilium sp. Highly reminiscent of anaerobic methane oxidation (AOM) consortia, these bacteria are spatially segregated within a planktonic macrostructure of extracellular polymeric substance in which they syntrophically couple sulfur oxidation and reduction reactions in a mutually beneficial arrangement that regenerates their respective sulfur substrates. As discussed here, the geochemical impacts of microbial metabolism are linked to the consortial organization and development of the pod structure, which affects cell-cell interactions and interactions with the surrounding geochemical microenvironment. If these pods are widespread in mine waters, echoing the now widespread discovery of AOM consortia, then AMD-driven CO(2) atmospheric fluxes from H(2)SO(4) carbonate weathering could be reduced by as much as 26 TgC/yr. This novel sulfur consortial discovery indicates that organized metabolically linked microbial partnerships are likely widespread and more significant in global elemental cycling than previously considered.

Isolation and characterization of ferrous- and sulfur-oxidizing bacteria from Tengchong solfataric region, China.

Microbial oxidation and reduction of iron and sulfur are important parts of biogeochemical cycles in acidic environments such as geothermal solfataric regions. Species of Acidithiobacillus and Leptospirillum are the common ferrous-iron and sulfur oxidizers from such environments. This study focused on the Tengchong sofataric region, located in Yunnan Province, Southwest China. Based on cultivation, 9 strains that grow on ferrous-iron and sulfuric compounds were obtained. Analysis of 16S rRNA genes of the 9 strains indicated that they were affiliated to Acidithiobacillus, Alicyclobacillus, Sulfobacillus, Leptospirillum and Acidiphilium. Physiological and phylogenetic studies indicated that two strains (TC-34 and TC-71) might represent two novel members of Alicyclobacillus. Strain TC-34 and TC-71 showed 94.8%-97.1% 16S rRNA gene identities to other species of Alicyclobacillus. Different from the previously described Alicyclobacillus species, strains TC-34 and TC-71 were mesophilic and their cellular fatty acids do not contain omega-cyclic fatty acids. Strain TC-71 was obligately dependent on ferrous-iron for growth. It was concluded that the ferrous-iron oxidizers were diversified and Alicyclobacillus species were proposed to take part in biochemical geocycling of iron in the Tengchong solfataric region.