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- Materials and methods
- Supporting Information
The Atacama Desert in northern Chile is a coastal desert usually described as one of the driest deserts on Earth, with a surface that has been minimally disturbed by natural erosion for millions of years (McKay et al. 2003; Hartley et al. 2005). This region includes arid and semi arid environments with many different saline deposits. In these saline deposits, evaporative basins called saltflats can be found (Chong 1984) containing a high percentage of salts from the leaching of volcanic rocks.
Until recently, regions of the Atacama Desert were considered the dry limit of photosynthetic activity and primary production (Warren-Rhodes et al. 2006), with extremely low levels of cultivable organisms and oxidized organic species detected in the soils (Navarro-González et al. 2003). In spite of the extreme conditions of this desert habitat, a broad spectrum of halotolerant and halophilic bacteria and archaeas have been found in saltflats and hypersaline lakes in the area (Demergasso et al. 2004; Wierzchos et al. 2006; Connon et al. 2007; De los Ríos et al. 2010).
Different classification schemes have been designed to define the relationships of micro-organisms with salt. The most widely accepted classification is based on the optimal salt concentration for their growth (Kushner and Kamekura 1988).
The halotolerance of many enzymes derived from halotolerant and halophilic micro-organisms can be exploited wherever enzymatic transformations are required to function under extreme physical and chemical conditions (Oren 2002a; Setati 2010). Hydrolases produced by halophiles have quite diverse potential usage in different areas (food industry, feed additive, biomedical sciences and chemical industries) (Rao et al. 1998; Kulkarni et al. 1999; Niehaus et al. 1999) and several halophilic enzymes have been tested for their potential biotechnological applications, including amylases, nucleases and proteases (Oren 2002b).
The Atacama Desert has increased its levels of heavy metals owing to natural and industrial processes (Salamanca et al. 2000). Cadmium, copper and zinc are among those heavy metals that are being released in the environment. Although some heavy metals constitute essential trace elements, most can be, at high concentrations, toxic to organisms from all branches of life, including bacteria (Nies 1999). However, some bacteria have adapted their physiology to tolerate the presence of metals or can even use them to grow. Owing to their exceptional characteristics, salt tolerant bacteria adapted to live in hostile environments may exhibit the potential to remove heavy metals from contaminated environments (Mishra et al. 2009; Zhuang et al. 2010). However, very little information is available on the heavy-metal tolerance of bacteria in hypersaline environments, with the exception of a few studies of metal-resistant halophilic bacteria isolated from different habitats such as the Maruit Lake, Egypt (Osman et al. 2010) or the Dead Sea Shore, Jordan (Amoozegar et al. 2005; Massadeh et al. 2005).
Therefore, in the present work, we studied the cultivable diversity of hydrolytic enzyme producers in a bacterial community from saline soils of the Atacama Desert. Furthermore, these strains were evaluated for their biotechnological potentials in terms of hydrolytic enzyme activity and heavy-metal tolerance.
- Top of page
- Materials and methods
- Supporting Information
Few environmental studies have focused on the study of the bacterial community inhabiting saline and hypersaline habitats in the Atacama Desert (Demergasso et al. 2004; Connon et al. 2007; Lester et al. 2007; Mishra et al. 2009; De los Ríos et al. 2010) and, in particular, to our knowledge, no screening programmes have been designed to select extremophilic micro-organisms with the ability to produce enzymes that hydrolyse polymeric compounds, although the hydrolases play a key role in the geochemical cycling of nutrients as the hydrolysis of high molecular weight biopolymers constitutes an initial step in the metabolism of organic compounds in the different ecosystems.
Under the restrictive conditions used in our screening, a total of 64 fresh cultures showing hydrolytic activities were isolated and from them, 25 strains were selected and characterized. Most isolates (16 strains) grow optimally in media containing between 5 and 7% (w/v) total salts, being able to grow in most cases up to 25% (w/v) salts. Thus, they can be classified as moderate halophiles according to the classification proposed by Kushner and Kamekura (1988). All the moderate halophiles were isolated from the AS. The number of halotolerant isolates was remarkably lower compared with that of moderate halophiles. Representative members of the halotolerant group were detected in both sampling sites, and the two extremely halotolerant strains (DV2 and AS2) were isolated from the Death Valley.
The most frequent hydrolytic activity detected in our study was DNase, followed by amylase and lipase activities. In the analysed community, we also detected pullulanase and protease producers. Xylanases were the least represented hydrolases. Sánchez-Porro et al. (2003) showed the abundance of five hydrolytic enzymes including amylase, protease, lipase, DNase and pullulanase in a community of moderate halophiles isolated from salterns in Spain, and they described amylase producers as the most abundant isolates. Furthermore, Moreno et al. (2009) studied the diversity of extreme halophiles, producing lipase, protease, amylase and nuclease in hypersaline ecosystems in South Spain, concluding that 70% of total of the hydrolytic isolates were also amylase producers. However, Baati et al. (2010) in the screening performed in salt mines of Sfax (Tunisia) detected a similar percentage of proteases, amylases and DNases producers. Rohban et al. (2009) investigated the ability of halophilic strains isolated from a hypersaline lake in Iran to produce nine different extracellular hydrolases (inulinase, pectinase, cellulase and xylanase). In contrast to our results, the most frequent activity expressed by these isolates was lipase, and the least prevalent activity represented was DNase.
It is interesting to emphasize that combined hydrolytic activity was frequently detected in isolates from both sampled areas, AS and the Death Valley. These results support previous studies in other hypersaline habitats (Sánchez-Porro et al. 2003; Moreno et al. 2009), although the number of strains showing combined hydrolytic activity is higher in the present study.
Based on the comparison of partial sequences of 16S rRNA genes, most environmental isolates able to produce hydrolytic enzymes were Gram-positive bacteria, assigned to the family Bacillaceae, comprising species of the genera Bacillus (Cohn 1872), Halobacillus (Spring et al. 1996) and Thalassobacillus (García et al. 2005). Bacillus is well known as an extracellular enzymes producer and many industrial processes use species of this genus for commercial production of enzymes (Schallmey et al. 2004). Only two isolates were related to the Gram-negative bacteria Ps. halophila (Sorokin et al. 2006) and H. organivorans (García et al. 2004). The other characterized isolates were related to S. roseus (Ventosa et al. 1990).
Although no organic matter is detected in the samples obtained from the AS, a broader diversity is found among the isolates if compared to that obtained from the Dead Valley, identifying only hydrolytic producers from the genus Bacillus. Probably, the presence of hydrolases is more significant among the population found in the AS samples to obtain nutrients in an adverse environment.
The cultivable microbial diversity found in this study is more limited than that detected in other ecological studies performed in diverse areas of the Atacama Desert, selecting only strains of the phyla γ-Proteobacteria (families Halomonadaceae and Pseudomonadaceae) and Firmicutes (families Bacillaceae and Staphylococcaceae). However, this study was carried out under more restrictive conditions. Our results differ from others in the preponderant hydrolytic bacteria. While the overall preponderant bacteria in our study were Firmicutes (92%), γ and β-Proteobacteria were reported as predominant in other studies (Rohban et al. 2009; Baati et al. 2010) in which Firmicutes did not appear or they were a minority.
Several studies have demonstrated that metal toxicity can be heavily influenced by environmental factors such as pH, temperature, soluble organic matter, clay minerals, inorganic anionic and cationic components (Nieto et al. 1989). Responses to the heavy metals tested in this study were very heterogeneous, with at least three different MICs being detected for the different metal ions in species belonging to the same genus (Table 3). In fact, four different MICs were found for zinc, copper and cobalt in Bacillus species. This suggests that halotolerant and halophilic bacteria express a very heterogeneous behaviour in connection with their individual natural susceptibility to the six heavy metals tested in the present study. Here, the highest toxicities were found with cadmium and zinc (80% of isolates were sensitive). This might be due to the fact that Cd is toxic per se and has not been found to be essential for biological functions in bacteria. Only one strain related to T. devorans showed high tolerance to cadmium (4·0 mmol l−1). The majority of strains were tolerant to nickel. The prevalence of Ni-resistant isolates has been shown in other saline environments previously described (Gaballa et al. 2003). Species related to H. hunanensis were the most sensitive to the heavy metals tested and the strains related to Bacillus subtilis and Bacillus stratosphericus were the most tolerant, showing tolerance to four different metal ions (Fe, Co, Ni and Cu) and moderate tolerance to Cd and Zn. Bacillus species have been shown to have the ability to accumulate gold, cadmium, chromium, copper, iron and manganese (El-meleigy et al. 2010). In our study, Gram-positive bacteria presented higher tolerance (less growth inhibition) to heavy metals than their Gram-negative counterparts, although Gram-negative bacteria tend to be the predominant prokaryotes in metal-polluted environments, owing to the structure and composition of their cell walls (Duxbury 1986).
In conclusion, in this work, we analysed and characterized the cultivable extremophilic hydrolytic bacterial community inhabiting the saltflats of the Atacama Desert. The isolated strains were evaluated for their biotechnological potentials in terms of hydrolytic enzyme activity and heavy-metal tolerance. We identify two outstanding implications: (i) the discovering of the Atacama Desert as a source for isolation of novel enzymes with biotechnological potential and (ii) development of heavy-metal-tolerant micro-organisms as potential innovative technology for bioremediation operations to remove heavy metal from saline soils.