Ecotoxicology (2014) 23:1922–1929 DOI 10.1007/s10646-014-1318-3
Response of growth and superoxide dismutase to enhanced arsenic in two Bacillus species Zuoming Xie • Xiaoyan Sun • Yanxin Wang Yan Luo • Xianjun Xie • Chunli Su
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Accepted: 9 August 2014 / Published online: 21 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Species differences in inorganic arsenic tolerance were investigated by comparing the responses of Bacillus subtilis (B. subtilis) and Bacillus thuringiensis (B. thuringiensis) to elevated concentrations of As(III) and As(V). The cell densities in treatments were always lower during the experiment compared to controls, with the exception of exposure to 1.0 mg As(V) l-1 on the first day. It was also found that relative growth rate (RGR) of B. thuringiensis was lower than that of B. subtilis. Furthermore, RGR of each Bacillus species was negative correlation with toxicity of inorganic arsenic. However, total cell number still increased in each treatment according to cell density and RGR assays. Superoxide dismutase (SOD) activity of both Bacillus species was promoted by As(III) and As(V), especially under high arsenic concentration condition. In addition, SOD activity of B. thuringiensis was higher than that of B. subtilis during the same exposure time. In lipid peroxidation assay, thiobarbituric acid-reactive substances (TBARS) content of each Bacillus species had a significant increase with increment of arsenic concentration. Moreover, significant difference was observed between the two Bacillus species under high arsenic concentration. TBARS content of B. thuringiensis was higher than that of B. subtilis, indicating that effect of arsenic on cell membranes of B. thuringiensis was much more than that of B. subtilis. These results suggest that the two Bacillus species could adapt and live in high arsenic aquifers, although their growth and cell membranes were affected by As treatment in a way.
Z. Xie X. Sun Y. Wang (&) Y. Luo X. Xie C. Su School of Environmental Studies, China University of Geosciences, Wuhan 430074, People’s Republic of China e-mail: [email protected]; [email protected]
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Keywords Arsenic High arsenic aquifer Superoxide dismutase Lipid peroxidation Bacillus
Introduction Arsenic is a toxic metalloid that is harmful to plants, animals, microorganisms and human beings (Liu et al. 2009). As(III) and As(V) are the two inorganic forms mainly present in high arsenic aquifers. Previous study has revealed that the former is generally found under reducing environments while the latter predominates under welloxidized conditions (Ackermann et al. 2008). However, in most cases, both As(III) and As(V) are found in either environment due to slow redox transformations and their different adsorption affinity (Bissen and Frimmel 2003; Corsini et al. 2010; Fitz and Wenzel 2002; Sadiq 1997). In addition, more and more studies demonstrated that microorganisms play a significant role in geochemical behaviour of arsenic (Bachate et al. 2009; Duan et al. 2009; Macur et al. 2001; Wang et al. 2010; Xie and Wang 2009; Xie et al. 2009a, 2011). As(III) can be oxidized to As(V) by chemoautotrophic arsenite oxidizer and heterotrophic arsenite oxidizer, or As(V) can be reduced to As(III) by dissimilatory arsenate-respiring prokaryote (Oremland and Stolz 2003; Sohrin et al. 1997). Hence, microbes behaviour can govern the changes in speciation of arsenic in shallow aquifers. On the contrary, the toxicity of arsenic also affects indigenous bacterial physiobiochemical characteristics. Organisms to long-term arsenic exposure have developed defence mechanisms. For instance, arsenic in organisms was detoxificated via several pathways, including reducing to arsenite, complexing with glutathione and phytochelatins, sequestrating into vacuoles, methylating to less toxic
Growth and superoxide dismutase of two Bacillus species under arsenic
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organic forms, or excluding from the cells (Rosen 1999; Xie 2008). Environmental stress including arsenic stress can increase levels of reactive oxygen species (ROS) in bacterial cells. Additionally, the high levels of ROS will disturb the steady-state balance of prooxidants and antioxidants. This unbalance in the oxidative metabolism of cells toward a prooxidant state is known as oxidative stress. Because SOD can catalyze the dismutation of O2 to H2O2 and O2, its resistance role to environmental stress has received much attention (Okamoto and Colepicolo 1998). Bacillus species are ubiquitous in nature, having been isolated from environments as diverse as freshwater, saline water, soil, plants, animals, and air (Pignatelli et al. 2009). Some Bacillus species by using arsenic as terminal electron acceptors can even grow in areas with high As level such as arsenic polluted soil (Cavalca et al. 2010), high arsenic shallow aquifers (Xie et al. 2011), as well as treatment plant waste (Vela´squez and Dussan 2009). Many studies have revealed that arsenic resistance (ars) gene clusters and arsenic accumulation of Bacillus species (Bhat et al. 2011; Joshi et al. 2009). However, few studies have been tried to elucidate the different effect of arsenic on superoxide dismutase (SOD) activities in indigenous Bacillus species in high arsenic aquifers. The objective of this present study was to assess the effects of inorganic arsenic on growth and SOD activity of two indigenous Bacillus species in microcosm experiments under laboratory conditions, so as to reveal the indigenous bacterial adaptation strategies to high arsenic stress in shallow aquifer system.
parameters in the present study. At Shuangzhai village, the typical range of arsenic concentration including As(III) and As(V) in the groundwater is 105.2–1,499 lg l-1 (Su 2006). Hence, the final concentrations of arsenic in the sterile media were set as 1.0 and 2.0 mg As(III) or As(V) l-1. And then both of the bacteria were cultured in the media under the above culture conditions. Furthermore, each treated group was prepared in triplicate and independently sampled simultaneously. The controls without arsenic were assayed in the same way as treatment samples.
Materials and methods
To compare the effects of high arsenic on the two bacterial growth, the relative growth rate (RGR: g dry mass increment per day per g biomass) was measured according to the method of Meerts and Garnier (1996) with slight modifications. Biomass of the samples was indicated with their optical density (OD) measured with spectrophotometer (Ultrospec 3000, Pharmacia Biotech Biochrom Ltd., England) at 640 nm. Hence, the unit of RGR was converted to day-1. RGR was constant during the first 10 days.
Biological material and culture conditions Bacillus subtilis (B. subtilis) and Bacillus thuringiensis (B. thuringiensis) were isolated from high arsenic aquifer sediments of Shuangzhai village (Shanyin County, Shanxi Province, China; 39°210 N, 112°510 E) in our previous works (Xie 2008). The organisms were cultivated in nutrient broth medium (1 % peptone, 0.3 % beef extract, and 0.5 % sodium chloride) at 25 ± 1 °C in darkness (Xie et al. 2011) to middle exponential phase for the following experiments. Arsenic treatments Both of the strains were cultured in nutrient broth medium with or without arsenic (As(III) or As(V)) respectively. Considering the environmental stress strength and exposure time are important influential factors to physiological and biochemical characteristics, they were used as main
Bacterial growth assays Cells inhibition tests Inhibition of arsenic on microbes was revealed by investigating the remaining ‘‘live’’ cells in the test samples. Samples were collected aseptically from the experimental groups at days 1, 2, 4, 7, and 10, respectively. To account for viable bacteria, each culture was aseptically diluted and then aliquots (0.1 ml) of dilution were uniformly spread on the sterile petri dishes with nutrient broth agar medium (nutrient broth medium with 2 % agar). All the agar plates were subsequently incubated under the above conditions for 7 days. Viable bacteria in each dilution were calculated according to the number of colony forming units (CFU) on the corresponding petri dishes. Therefore, the number of CFU multiplied by the dilution factor was the total ‘‘live’’ cells in each sample. Relative growth rate measurements
Measurements of superoxide dismutase For enzyme activity assessment, the bacterial cells were collected from 400 ml of bacterial culture after 2 days of incubation. The cells were resuspended in 1 ml of Tris/ borate (0.1 M/0.3 M, pH 7.5, 5 mM EDTA, and 7 nM b-mercaptoethanol) buffer on ice for 10 min. Debris was removed from the extract by centrifugation at 10,0009g for 20 min at 4 °C. The supernatant was used as crude extract for SOD activity assay (Kong and Sang 1999).
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SOD activity was determined by pyrogallol autoxidation test as described by Marklund and Marklund (1974). Protein concentrations in the two bacterial cells were determined by the method of Bradford (1976) using bovine serum albumin as a standard protein. Enzyme activity was expressed in U mg-1 soluble protein. Lipid peroxidation assay Lipid damage was estimated through thiobarbituric acidreactive substances (TBARS) content by the procedure of Semchyshyn et al. (2005) with slightly modification. For the measurement, 5 ml of bacterial culture after 2 days of incubation was centrifuged at 4,0009g for 10 min. The cell pellet in 5 ml of 0.1 % TCA was homogenized for 30 s with a sonicator in ice bath. Equal volumes of homogenate and 0.5 % thiobarbituric acid (TBA) in 20 % (w:v) trichloroacetic acid (TCA) solution were mixed and incubated at 100 °C for 60 min in a water bath. The mixture was quickly cooled in an ice-bath and then centrifuged at 10,0009g for 10 min. The absorbance of the supernatant was recorded at 535 nm with spectrophotometry. TBARS content was determined using an extinction coefficient 156 mM-1 cm-1. The result was expressed as nmol per mg protein. Statistical analysis Data were analyzed using one-way ANOVA test as available in the SPSS statistical package with a significance level of p \ 0.05. All figures were described by PC-based Origin program.
Results Growth of two Bacillus species Cell densities of two Bacillus species in the media added with As(III) and 2.0 mg As(V) l-1 had a decrease on the first day, subsequently increased gradually. However, CFU of the culture exposed to 1.0 mg As(V) l-1 had an increase even beyond the control on the first day. The amount of total ‘‘live’’ cells of the two Bacillus species increased in each experimental group at high arsenic level after 10 days of incubation, but they still did not overtake the initial value in the treated groups exposed to 2.0 mg As l-1 except for B. subtilis in the media with As(V). Nevertheless, cell viability in treatment groups compared to control was lower at the same culture time, with the exception of exposure to the low concentration of As(V) on the first day (Fig. 1). In addition, the cell density of the same bacterial species was also lower exposure to As(III) than exposure to
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As(V) at the same culture period. It was also shown in Fig. 1 that cell viability of the same Bacillus species in treatment groups exposed to As(V) was higher than that exposed to As(III). From Fig. 2, it was found that RGR between the two tested Bacillus strains was obviously different under the same inorganic arsenic condition. RGR of B. subtilis was higher than that of B. thuringiensis whether exposure to As(III) or As(V). Figure 2 also indicated that RGR of each Bacillus species had a significant variation between the different As media. Furthermore, RGR in the control of each Bacillus species was higher than either exposed to As(III) or As(V), however, a little difference of RGR between the control and the treatment exposure to 1.0 mg As(V) l-1 was shown in the Fig. 2b. In addition, RGR in the media supplemented with As(III) was lower than that added with As(V) (Fig. 2). Activities of SOD activities and content of TBARS As shown in Fig. 3, the SOD activities of B. thuringiensis were higher than those of B. subtilis except the treatment exposure to 1.0 mg As(III) l-1. By contrast, they had a little difference between the two Bacillus under low As environments. The SOD activities of both B. subtilis and B. thuringiensis increased significantly (p \ 0.05) with increase of concentration of As(III) or As(V). Moreover, there was a significant variation between exposed to As(III) and As(V), the bacterial cells in the media added with As(III) showing higher values of SOD activities. TBARS contents in B. thuringiensis were higher significantly (p \ 0.05) than those in B. subtilis at high arsenic levels, while TBARS contents in the two Bacillus species had little difference at low arsenic levels. In addition, TBARS contents in B. thuringiensis or B. subtilis exposed to As(III) were higher than those exposed to As(V) at the same arsenic level (Fig. 4).
Discussion Impact of arsenic on growth According to the results of the CFU count assay, the viability of the two Bacillus species was significantly reduced when arsenic was added to the media. This result can be explained by the toxicity of As. Arsenic toxicity for organisms is well known, not only for humans, animals and plants but also for microbes (Fowler 1983; Pisani et al. 2011). As(III) is extremely toxic to microbes (Huysmans and Frankenberger 1990), and its toxicity is much more violent to organisms than any other As species (Oremland and Stolz 2005). In the present study, the cell viability had
Growth and superoxide dismutase of two Bacillus species under arsenic
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Fig. 1 Changes of cell density of two Bacillus species in the culture exposed to As(III) or As(V). a, c B. subtilis; b, d B. thuringiensis. Each data point is the mean ± SE. *p \ 0.05, here *significantly different from controls on the same date
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Fig. 2 Influence of different inorganic arsenic on the RGR of two Bacillus species. a As(III); b As(V). Each data point is the mean ± SE. *p \ 0.05, here *significantly different from controls on the same date
a reduction at the beginning of exposure to As(III) and high As(V) level, which was compatible with the results of Fulladosa et al. (2005) who reported the viability of
bacterium had a decrease during the first hours after treated with arsenic. Nagy et al. (2005) also pointed out that the density and volume of cyanobacteria was reduced in
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Fig. 3 Dose-dependent induction of SOD activity in cells of two Bacillus species after 2 days exposure to different inorganic arsenic. a As(III); b As(V). Each data point is the mean ± SE. *p \ 0.05, here *significantly different from controls on the same date
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Fig. 4 Effect of different inorganic arsenic on TBARS contents in cells of two Bacillus species after 2 days of incubation. a As(III); b As(V). Each data point is the mean ± SE. *p \ 0.05, here *significantly different from controls on the same date
As-contaminated areas. It was also observed in the present study that cell density began to increase after 2 days of exposure to As, suggesting that the two Bacillus could have adapted the limiting conditions and withstand the deleterious effects of arsenic. Previous studies also showed that bacteria can adapt elevated concentrations of arsenic (Cervantes et al. 1994; de Groot et al. 2003; Shilev et al. 2007). The cell densities of the two Bacillus species in the treatments supplemented with As(V) were low compared to control, except with low As(V) level in the first day, indicating As(V) was toxic for Bacillus and affected their cellular reproduce. The toxicity of arsenate to organisms is due to its action as a phosphate analogue, which resulted in a mistake that arsenate was transported into cells as phosphate via phosphate cotransport systems (Juma and
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Tabatabai 1977; Speir et al. 1999; Ullrich-Eberius et al. 1989). Thus, the main toxicity of arsenate results from its interference with the metabolism of the major bioelement phosphorus (Nies 1999). In addition, because of the high phosphate concentration in the cell, the structural similarity makes it difficult to export arsenate effectively out from cells after arsenate has been taken up by phosphate transport systems (Nies and Silver 1995). Hence, the reason for a decline of cell density in the media may be that metabolism of Bacillus was inhibited by elevated arsenic content in bacterial cells. The abundance of Bacillus confirmed the strong resistance of this genus to conditions of stress (Ellis et al. 2003). The number of viable bacteria and RGR of B. subtilis were higher than those of B. thuringiensis exposure to the same speciation and concentration of arsenic, suggesting that
Growth and superoxide dismutase of two Bacillus species under arsenic
B. subtilis exhibited higher tolerance to As compared to B. thuringiensis. This can be explained by toxic difference. Arsenic toxicity is variable for different bacteria (Hallberg et al. 1996; Takeuchi et al. 2007). Hence, although the initial biomass of B. thuringiensis in per volume was much more than that of B. subtilis, the total ‘‘live’’ cells in the treatments of the former was lower than those of the later. Microbes in high arsenic environments have the mechanisms conferring tolerance to contain arsenic compounds (Li et al. 2010; Xie 2008). These mechanisms include detoxification through arsenic resistance system (Bruhn et al. 1996; Kloppers et al. 2008), alterations in transmembrane transportation to minimize arsenate uptake, arsenic oxidation or reduction systems (Anderson et al. 1992; Sanders and Windom 1980), arsenic methylation pathways (Anderson et al. 1992), and arsenic sequestration via multidrug resistance associated proteins (Ghosh et al. 1999; Li et al. 2010). Therefore, the arsenic resistance mechanisms or ability in B. subtilis could be distinct from that in B. thuringiensis.
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activity in B. thuringiensis was higher than that in B. subtilis under the same stress, suggesting that the response of B. thuringiensis to toxicity of arsenic was more violent than that of B. subtilis. Therefore, the cell of B. thuringiensis was easy to be damaged by inorganic arsenic compared to B. subtilis. With an increase of SOD activity under arsenic treatment, Bacillus seems well protected against the potential threat of oxidative stress arising under enhanced arsenic. Hence, indigenous Bacillus species could highly adapt to arsenic stress and live in high arsenic aquifers. TBARS contents as SOD activity in the two Bacillus species increased significantly (p \ 0.05) compared with controls and ascended with increment of As concentration, indicating that cell membranes could have been affected. Because As enhances lipid peroxidation by disorganizing the membrane structure (Obermu¨ller et al. 2005). Cell membrane damage is an important cause of viability reduction. Hence, the results of membrane lipid peroxidation can also demonstrated the above conclusion that B. thuringiensis exposed to As was more vulnerable compared to B. subtilis.
Effect of arsenic on SOD and TBARS ROS is produced in cells exposed to environmental stresses such as metals, UV-radiation, high light intensities, drought, salt, heat and freezing (Xie et al. 2009b). The high level of ROS is potentially harmful to the normal metabolism and results in oxidative injury including lipid peroxidation, protein degradation, as well as DNA damage (He and Ha¨der 2002; Jordan 1996). Furthermore, cell damage resulted from these stresses can be mediated by oxidative stress. Hence, the induction of antioxidant enzymes like SOD is an important protective mechanism to minimize cell oxidative damage in environmental stresses (Okamoto and Colepicolo 1998). Since SOD is one of the key enzymes to eliminate active oxygen (Kong and Sang 1999), and SOD as important antioxidant enzyme was found in almost all organisms, SOD has been regarded as a probe of oxidative stress in many studies (Davidson and Pearson 1996; Okamoto and Colepicolo 1998; Perl-Treves and Galun 1991; Tsang et al. 1991; Xie et al. 2009b). The results in this experiments also showed that arsenic resulted in the significant increase of SOD activities compared with the control (p \ 0.05), and SOD activities enhanced with the increment of concentrations and toxicity of arsenic. It was well known that well-developed antioxidant defence mechanisms scavenge ROS to establish a balance between pro-oxidant and antioxidant processes before critical concentrations build up under non-stressed conditions (Obermu¨ller et al. 2005). However, once the toxicity of arsenic to cells increased, the balance would be upset, and then the cellular detoxification system was stimulated and the synthesis of SOD was started. It was also observed that SOD
Conclusion According to the growth and SOD activity in the two Bacillus species exposure to As tested in this present study, it can be summarized as follows: B. subtilis exhibited higher tolerance to As compared to B. thuringiensis. A slight effect of low As(V) level on the growth of the two Bacillus species was observed. However, the growth was significantly restrained under the conditions of As(III) and high As(V) level. SOD activities of the two Bacillus species were promoted when exposed to inorganic arsenic. SOD should play an important role to prevent from the potential threat of oxidative stress arising under enhanced arsenic. Acknowledgments The research work was financially supported by the National Natural Science Foundation of China (Grant Nos. 41172219, 41120124003, 40830748, 40425001 and 40830748), the Special Fund for Basic Scientific Research of Central Colleges, China University of Geosciences (Wuhan) (CUGL090220), Ministry of Science and Technology of China (2008DFA20950) and the 111 project (B08030). Conflict of interest of interest.
The authors declare that they have no conflict
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Response of growth and superoxide dismutase to enhanced arsenic in two Bacillus species Zuoming Xie • Xiaoyan Sun • Yanxin Wang Yan Luo • Xianjun Xie • Chunli Su
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Accepted: 9 August 2014 / Published online: 21 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Species differences in inorganic arsenic tolerance were investigated by comparing the responses of Bacillus subtilis (B. subtilis) and Bacillus thuringiensis (B. thuringiensis) to elevated concentrations of As(III) and As(V). The cell densities in treatments were always lower during the experiment compared to controls, with the exception of exposure to 1.0 mg As(V) l-1 on the first day. It was also found that relative growth rate (RGR) of B. thuringiensis was lower than that of B. subtilis. Furthermore, RGR of each Bacillus species was negative correlation with toxicity of inorganic arsenic. However, total cell number still increased in each treatment according to cell density and RGR assays. Superoxide dismutase (SOD) activity of both Bacillus species was promoted by As(III) and As(V), especially under high arsenic concentration condition. In addition, SOD activity of B. thuringiensis was higher than that of B. subtilis during the same exposure time. In lipid peroxidation assay, thiobarbituric acid-reactive substances (TBARS) content of each Bacillus species had a significant increase with increment of arsenic concentration. Moreover, significant difference was observed between the two Bacillus species under high arsenic concentration. TBARS content of B. thuringiensis was higher than that of B. subtilis, indicating that effect of arsenic on cell membranes of B. thuringiensis was much more than that of B. subtilis. These results suggest that the two Bacillus species could adapt and live in high arsenic aquifers, although their growth and cell membranes were affected by As treatment in a way.
Z. Xie X. Sun Y. Wang (&) Y. Luo X. Xie C. Su School of Environmental Studies, China University of Geosciences, Wuhan 430074, People’s Republic of China e-mail: [email protected]; [email protected]
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Keywords Arsenic High arsenic aquifer Superoxide dismutase Lipid peroxidation Bacillus
Introduction Arsenic is a toxic metalloid that is harmful to plants, animals, microorganisms and human beings (Liu et al. 2009). As(III) and As(V) are the two inorganic forms mainly present in high arsenic aquifers. Previous study has revealed that the former is generally found under reducing environments while the latter predominates under welloxidized conditions (Ackermann et al. 2008). However, in most cases, both As(III) and As(V) are found in either environment due to slow redox transformations and their different adsorption affinity (Bissen and Frimmel 2003; Corsini et al. 2010; Fitz and Wenzel 2002; Sadiq 1997). In addition, more and more studies demonstrated that microorganisms play a significant role in geochemical behaviour of arsenic (Bachate et al. 2009; Duan et al. 2009; Macur et al. 2001; Wang et al. 2010; Xie and Wang 2009; Xie et al. 2009a, 2011). As(III) can be oxidized to As(V) by chemoautotrophic arsenite oxidizer and heterotrophic arsenite oxidizer, or As(V) can be reduced to As(III) by dissimilatory arsenate-respiring prokaryote (Oremland and Stolz 2003; Sohrin et al. 1997). Hence, microbes behaviour can govern the changes in speciation of arsenic in shallow aquifers. On the contrary, the toxicity of arsenic also affects indigenous bacterial physiobiochemical characteristics. Organisms to long-term arsenic exposure have developed defence mechanisms. For instance, arsenic in organisms was detoxificated via several pathways, including reducing to arsenite, complexing with glutathione and phytochelatins, sequestrating into vacuoles, methylating to less toxic
Growth and superoxide dismutase of two Bacillus species under arsenic
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organic forms, or excluding from the cells (Rosen 1999; Xie 2008). Environmental stress including arsenic stress can increase levels of reactive oxygen species (ROS) in bacterial cells. Additionally, the high levels of ROS will disturb the steady-state balance of prooxidants and antioxidants. This unbalance in the oxidative metabolism of cells toward a prooxidant state is known as oxidative stress. Because SOD can catalyze the dismutation of O2 to H2O2 and O2, its resistance role to environmental stress has received much attention (Okamoto and Colepicolo 1998). Bacillus species are ubiquitous in nature, having been isolated from environments as diverse as freshwater, saline water, soil, plants, animals, and air (Pignatelli et al. 2009). Some Bacillus species by using arsenic as terminal electron acceptors can even grow in areas with high As level such as arsenic polluted soil (Cavalca et al. 2010), high arsenic shallow aquifers (Xie et al. 2011), as well as treatment plant waste (Vela´squez and Dussan 2009). Many studies have revealed that arsenic resistance (ars) gene clusters and arsenic accumulation of Bacillus species (Bhat et al. 2011; Joshi et al. 2009). However, few studies have been tried to elucidate the different effect of arsenic on superoxide dismutase (SOD) activities in indigenous Bacillus species in high arsenic aquifers. The objective of this present study was to assess the effects of inorganic arsenic on growth and SOD activity of two indigenous Bacillus species in microcosm experiments under laboratory conditions, so as to reveal the indigenous bacterial adaptation strategies to high arsenic stress in shallow aquifer system.
parameters in the present study. At Shuangzhai village, the typical range of arsenic concentration including As(III) and As(V) in the groundwater is 105.2–1,499 lg l-1 (Su 2006). Hence, the final concentrations of arsenic in the sterile media were set as 1.0 and 2.0 mg As(III) or As(V) l-1. And then both of the bacteria were cultured in the media under the above culture conditions. Furthermore, each treated group was prepared in triplicate and independently sampled simultaneously. The controls without arsenic were assayed in the same way as treatment samples.
Materials and methods
To compare the effects of high arsenic on the two bacterial growth, the relative growth rate (RGR: g dry mass increment per day per g biomass) was measured according to the method of Meerts and Garnier (1996) with slight modifications. Biomass of the samples was indicated with their optical density (OD) measured with spectrophotometer (Ultrospec 3000, Pharmacia Biotech Biochrom Ltd., England) at 640 nm. Hence, the unit of RGR was converted to day-1. RGR was constant during the first 10 days.
Biological material and culture conditions Bacillus subtilis (B. subtilis) and Bacillus thuringiensis (B. thuringiensis) were isolated from high arsenic aquifer sediments of Shuangzhai village (Shanyin County, Shanxi Province, China; 39°210 N, 112°510 E) in our previous works (Xie 2008). The organisms were cultivated in nutrient broth medium (1 % peptone, 0.3 % beef extract, and 0.5 % sodium chloride) at 25 ± 1 °C in darkness (Xie et al. 2011) to middle exponential phase for the following experiments. Arsenic treatments Both of the strains were cultured in nutrient broth medium with or without arsenic (As(III) or As(V)) respectively. Considering the environmental stress strength and exposure time are important influential factors to physiological and biochemical characteristics, they were used as main
Bacterial growth assays Cells inhibition tests Inhibition of arsenic on microbes was revealed by investigating the remaining ‘‘live’’ cells in the test samples. Samples were collected aseptically from the experimental groups at days 1, 2, 4, 7, and 10, respectively. To account for viable bacteria, each culture was aseptically diluted and then aliquots (0.1 ml) of dilution were uniformly spread on the sterile petri dishes with nutrient broth agar medium (nutrient broth medium with 2 % agar). All the agar plates were subsequently incubated under the above conditions for 7 days. Viable bacteria in each dilution were calculated according to the number of colony forming units (CFU) on the corresponding petri dishes. Therefore, the number of CFU multiplied by the dilution factor was the total ‘‘live’’ cells in each sample. Relative growth rate measurements
Measurements of superoxide dismutase For enzyme activity assessment, the bacterial cells were collected from 400 ml of bacterial culture after 2 days of incubation. The cells were resuspended in 1 ml of Tris/ borate (0.1 M/0.3 M, pH 7.5, 5 mM EDTA, and 7 nM b-mercaptoethanol) buffer on ice for 10 min. Debris was removed from the extract by centrifugation at 10,0009g for 20 min at 4 °C. The supernatant was used as crude extract for SOD activity assay (Kong and Sang 1999).
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SOD activity was determined by pyrogallol autoxidation test as described by Marklund and Marklund (1974). Protein concentrations in the two bacterial cells were determined by the method of Bradford (1976) using bovine serum albumin as a standard protein. Enzyme activity was expressed in U mg-1 soluble protein. Lipid peroxidation assay Lipid damage was estimated through thiobarbituric acidreactive substances (TBARS) content by the procedure of Semchyshyn et al. (2005) with slightly modification. For the measurement, 5 ml of bacterial culture after 2 days of incubation was centrifuged at 4,0009g for 10 min. The cell pellet in 5 ml of 0.1 % TCA was homogenized for 30 s with a sonicator in ice bath. Equal volumes of homogenate and 0.5 % thiobarbituric acid (TBA) in 20 % (w:v) trichloroacetic acid (TCA) solution were mixed and incubated at 100 °C for 60 min in a water bath. The mixture was quickly cooled in an ice-bath and then centrifuged at 10,0009g for 10 min. The absorbance of the supernatant was recorded at 535 nm with spectrophotometry. TBARS content was determined using an extinction coefficient 156 mM-1 cm-1. The result was expressed as nmol per mg protein. Statistical analysis Data were analyzed using one-way ANOVA test as available in the SPSS statistical package with a significance level of p \ 0.05. All figures were described by PC-based Origin program.
Results Growth of two Bacillus species Cell densities of two Bacillus species in the media added with As(III) and 2.0 mg As(V) l-1 had a decrease on the first day, subsequently increased gradually. However, CFU of the culture exposed to 1.0 mg As(V) l-1 had an increase even beyond the control on the first day. The amount of total ‘‘live’’ cells of the two Bacillus species increased in each experimental group at high arsenic level after 10 days of incubation, but they still did not overtake the initial value in the treated groups exposed to 2.0 mg As l-1 except for B. subtilis in the media with As(V). Nevertheless, cell viability in treatment groups compared to control was lower at the same culture time, with the exception of exposure to the low concentration of As(V) on the first day (Fig. 1). In addition, the cell density of the same bacterial species was also lower exposure to As(III) than exposure to
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As(V) at the same culture period. It was also shown in Fig. 1 that cell viability of the same Bacillus species in treatment groups exposed to As(V) was higher than that exposed to As(III). From Fig. 2, it was found that RGR between the two tested Bacillus strains was obviously different under the same inorganic arsenic condition. RGR of B. subtilis was higher than that of B. thuringiensis whether exposure to As(III) or As(V). Figure 2 also indicated that RGR of each Bacillus species had a significant variation between the different As media. Furthermore, RGR in the control of each Bacillus species was higher than either exposed to As(III) or As(V), however, a little difference of RGR between the control and the treatment exposure to 1.0 mg As(V) l-1 was shown in the Fig. 2b. In addition, RGR in the media supplemented with As(III) was lower than that added with As(V) (Fig. 2). Activities of SOD activities and content of TBARS As shown in Fig. 3, the SOD activities of B. thuringiensis were higher than those of B. subtilis except the treatment exposure to 1.0 mg As(III) l-1. By contrast, they had a little difference between the two Bacillus under low As environments. The SOD activities of both B. subtilis and B. thuringiensis increased significantly (p \ 0.05) with increase of concentration of As(III) or As(V). Moreover, there was a significant variation between exposed to As(III) and As(V), the bacterial cells in the media added with As(III) showing higher values of SOD activities. TBARS contents in B. thuringiensis were higher significantly (p \ 0.05) than those in B. subtilis at high arsenic levels, while TBARS contents in the two Bacillus species had little difference at low arsenic levels. In addition, TBARS contents in B. thuringiensis or B. subtilis exposed to As(III) were higher than those exposed to As(V) at the same arsenic level (Fig. 4).
Discussion Impact of arsenic on growth According to the results of the CFU count assay, the viability of the two Bacillus species was significantly reduced when arsenic was added to the media. This result can be explained by the toxicity of As. Arsenic toxicity for organisms is well known, not only for humans, animals and plants but also for microbes (Fowler 1983; Pisani et al. 2011). As(III) is extremely toxic to microbes (Huysmans and Frankenberger 1990), and its toxicity is much more violent to organisms than any other As species (Oremland and Stolz 2005). In the present study, the cell viability had
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a reduction at the beginning of exposure to As(III) and high As(V) level, which was compatible with the results of Fulladosa et al. (2005) who reported the viability of
bacterium had a decrease during the first hours after treated with arsenic. Nagy et al. (2005) also pointed out that the density and volume of cyanobacteria was reduced in
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As-contaminated areas. It was also observed in the present study that cell density began to increase after 2 days of exposure to As, suggesting that the two Bacillus could have adapted the limiting conditions and withstand the deleterious effects of arsenic. Previous studies also showed that bacteria can adapt elevated concentrations of arsenic (Cervantes et al. 1994; de Groot et al. 2003; Shilev et al. 2007). The cell densities of the two Bacillus species in the treatments supplemented with As(V) were low compared to control, except with low As(V) level in the first day, indicating As(V) was toxic for Bacillus and affected their cellular reproduce. The toxicity of arsenate to organisms is due to its action as a phosphate analogue, which resulted in a mistake that arsenate was transported into cells as phosphate via phosphate cotransport systems (Juma and
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Tabatabai 1977; Speir et al. 1999; Ullrich-Eberius et al. 1989). Thus, the main toxicity of arsenate results from its interference with the metabolism of the major bioelement phosphorus (Nies 1999). In addition, because of the high phosphate concentration in the cell, the structural similarity makes it difficult to export arsenate effectively out from cells after arsenate has been taken up by phosphate transport systems (Nies and Silver 1995). Hence, the reason for a decline of cell density in the media may be that metabolism of Bacillus was inhibited by elevated arsenic content in bacterial cells. The abundance of Bacillus confirmed the strong resistance of this genus to conditions of stress (Ellis et al. 2003). The number of viable bacteria and RGR of B. subtilis were higher than those of B. thuringiensis exposure to the same speciation and concentration of arsenic, suggesting that
Growth and superoxide dismutase of two Bacillus species under arsenic
B. subtilis exhibited higher tolerance to As compared to B. thuringiensis. This can be explained by toxic difference. Arsenic toxicity is variable for different bacteria (Hallberg et al. 1996; Takeuchi et al. 2007). Hence, although the initial biomass of B. thuringiensis in per volume was much more than that of B. subtilis, the total ‘‘live’’ cells in the treatments of the former was lower than those of the later. Microbes in high arsenic environments have the mechanisms conferring tolerance to contain arsenic compounds (Li et al. 2010; Xie 2008). These mechanisms include detoxification through arsenic resistance system (Bruhn et al. 1996; Kloppers et al. 2008), alterations in transmembrane transportation to minimize arsenate uptake, arsenic oxidation or reduction systems (Anderson et al. 1992; Sanders and Windom 1980), arsenic methylation pathways (Anderson et al. 1992), and arsenic sequestration via multidrug resistance associated proteins (Ghosh et al. 1999; Li et al. 2010). Therefore, the arsenic resistance mechanisms or ability in B. subtilis could be distinct from that in B. thuringiensis.
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activity in B. thuringiensis was higher than that in B. subtilis under the same stress, suggesting that the response of B. thuringiensis to toxicity of arsenic was more violent than that of B. subtilis. Therefore, the cell of B. thuringiensis was easy to be damaged by inorganic arsenic compared to B. subtilis. With an increase of SOD activity under arsenic treatment, Bacillus seems well protected against the potential threat of oxidative stress arising under enhanced arsenic. Hence, indigenous Bacillus species could highly adapt to arsenic stress and live in high arsenic aquifers. TBARS contents as SOD activity in the two Bacillus species increased significantly (p \ 0.05) compared with controls and ascended with increment of As concentration, indicating that cell membranes could have been affected. Because As enhances lipid peroxidation by disorganizing the membrane structure (Obermu¨ller et al. 2005). Cell membrane damage is an important cause of viability reduction. Hence, the results of membrane lipid peroxidation can also demonstrated the above conclusion that B. thuringiensis exposed to As was more vulnerable compared to B. subtilis.
Effect of arsenic on SOD and TBARS ROS is produced in cells exposed to environmental stresses such as metals, UV-radiation, high light intensities, drought, salt, heat and freezing (Xie et al. 2009b). The high level of ROS is potentially harmful to the normal metabolism and results in oxidative injury including lipid peroxidation, protein degradation, as well as DNA damage (He and Ha¨der 2002; Jordan 1996). Furthermore, cell damage resulted from these stresses can be mediated by oxidative stress. Hence, the induction of antioxidant enzymes like SOD is an important protective mechanism to minimize cell oxidative damage in environmental stresses (Okamoto and Colepicolo 1998). Since SOD is one of the key enzymes to eliminate active oxygen (Kong and Sang 1999), and SOD as important antioxidant enzyme was found in almost all organisms, SOD has been regarded as a probe of oxidative stress in many studies (Davidson and Pearson 1996; Okamoto and Colepicolo 1998; Perl-Treves and Galun 1991; Tsang et al. 1991; Xie et al. 2009b). The results in this experiments also showed that arsenic resulted in the significant increase of SOD activities compared with the control (p \ 0.05), and SOD activities enhanced with the increment of concentrations and toxicity of arsenic. It was well known that well-developed antioxidant defence mechanisms scavenge ROS to establish a balance between pro-oxidant and antioxidant processes before critical concentrations build up under non-stressed conditions (Obermu¨ller et al. 2005). However, once the toxicity of arsenic to cells increased, the balance would be upset, and then the cellular detoxification system was stimulated and the synthesis of SOD was started. It was also observed that SOD
Conclusion According to the growth and SOD activity in the two Bacillus species exposure to As tested in this present study, it can be summarized as follows: B. subtilis exhibited higher tolerance to As compared to B. thuringiensis. A slight effect of low As(V) level on the growth of the two Bacillus species was observed. However, the growth was significantly restrained under the conditions of As(III) and high As(V) level. SOD activities of the two Bacillus species were promoted when exposed to inorganic arsenic. SOD should play an important role to prevent from the potential threat of oxidative stress arising under enhanced arsenic. Acknowledgments The research work was financially supported by the National Natural Science Foundation of China (Grant Nos. 41172219, 41120124003, 40830748, 40425001 and 40830748), the Special Fund for Basic Scientific Research of Central Colleges, China University of Geosciences (Wuhan) (CUGL090220), Ministry of Science and Technology of China (2008DFA20950) and the 111 project (B08030). Conflict of interest of interest.
The authors declare that they have no conflict
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