Abstract: Chromium (VI) contamination has accelerated due to rapid industrialization worldwide. Aim of this study is to check the bacterial species for their tolerance towards multiple metals, antibiotics and plant growth promoting activity and further check whether these bacteria are reducing Cr (VI). Bacterial strains were isolated from metal contaminated soils of Abeokuta. All of the isolates showed tolerance to lead, zinc and chromium (VI). Bacterial specie also showed tolerance towards antibiotics, 100% of the isolates were tolerant to Septrin, Chloramphenicol, Sparfloxacin, Amoxicillin, Augmentin, Tarivid and Streptomycin, whereas 83.33% were tolerant to Gentamycin and Pefloxacin and 33.33% were resistant to Ciprofloxacin. All bacterial species were positive to ammonia, whereas strain PZ3 and PZ4 were found to be positive to HCN. Among all the strains, only Streptococcus spp. PZ4 showed reduction of Chromium (VI). Maximum reduction (85%) of chromium (VI) was observed at pH 7 by Streptococcus spp. PZ4. Similarly, Streptococcus spp. PZ4 reduced the chromium considerably at pH 5 (51.25%), pH 6 (72.5%), pH 8 (67.5%) and at pH 9 (45%), at a concentration of 100 μg Cr mL-1 after 120 h of incubation. Streptococcus spp. PZ4 reduced chromium (VI) at a concentration of 50 μg Cr mL-1 (47.5%), 100 μg Cr mL-1 (91.25%) and 150 μg Cr mL-1 (134.17%). Due to above properties strains could therefore be used as bioremediators of metals in soils contaminated with heavy metals and can also increase the yield of various crops under heavy metal contamination.
INTRODUCTION
The contamination of chromium (VI) mainly due to the use of Cr (VI) in leather, tanning, metallurgy, electroplating, textile and pigment manufacturing industries (Wang and Xiao, 1995; Pattanapipitpaisal et al., 2001; Sultan and Hasnain, 2007). Chromium occurs either in trivalent or hexavalent which affect growth of microorganisms present in the environment (Ortegel et al., 2002). Hexavalent chromium is highly soluble in water, permeable through biological membranes and it interacts with proteins and nucleic acids which makes it more toxic and carcinogenic than trivalent (Kamaludeen et al., 2003; Ackerley et al., 2006). Reduction of Cr (VI) leads to the formation of stables, less soluble and less toxic Cr (III). Reduction of toxic Cr (VI) to Cr (III) is thus a useful process for remediation of Cr (VI) affected environments (Jeyasingh and Phillip, 2005). The reduction/detoxification of Cr (VI) by microbes is, however, inexpensive and environmentally safe approach and provides a viable option to protect the environment from chromium toxicity. The reduction of Cr (VI) has been reported in Bacillus sp. (Elangovan et al., 2006; Chaturvedi, 2011), Pseudomonas sp. (Rahman et al., 2007), Escherichia coli (Bae et al., 2005), Microbacterium (Pattanapipitpaisal et al., 2001), Ochrobactrum intermedium (Faisal and Hasnain, 2005) and Micrococcus (Sultan and Hasnain, 2005).
Detoxification of chromium by microbes may occur directly or indirectly and is affected by pH, chromate concentration, incubation periods and the types of microbes involved. In the direct mechanism microbes enzymatically (chromium reductases) reduce chromium (Losi et al., 1994) while in the indirect method, reductants or oxidant, such as H2S, reduce chromium (DeFilippi and Lupton, 1992). Furthermore, in growing cultures with added carbon sources as electron donors and in cell suspensions, Cr (VI) reduction can be predominantly aerobic or anaerobic but generally not both. Interestingly, chromium reductases can catalyse reduction of Cr (VI) to Cr (III) anaerobically (Lovley and Coates, 1997), aerobically (Cervantes et al., 2001) and also both anaerobically and aerobically (Marsh and McInerney, 2001). The Cr (VI) reductase may be present in the membrane fraction of the cells of PGPR, as found in Pseudomonas fluorescens and Enterobacter cloacae (Wang et al., 1990). Further evidence suggested that cytochrome c548 was involved in the reduction of Cr (VI) by membrane vesicles. In the presence of H2 and excess of hydrogenase, cytochrome C3, a periplasmic protein, in the soluble cell free fraction of D. vulgaris (Lovley and Coates, 1997), reduced Cr (VI), 50 times faster than did the Cr (VI) reductase of P. ambigua with NADH and NADPH, as electron donor (Horitsu et al., 1987). Under anaerobic condition Cr (VI) reduction is due to the action of enzymes associated with membranes of the electron transfer system (Cervantes and Campos, 2007). The reduction to Cr (III) results in the formation of insoluble precipitate [Cr(OH)3] which is easily removed from wastewater (Jeyasingh and Phillip, 2005). The enzyme chromium reductase found in P. ambigua (Campos-Garcia et al., 1997) and Bacillus sp. (Wang et al., 1991) were purified and characterized. More recently, to clone a chromate reductase gene, novel soluble chromate reductase of P. putida was purified to homogeneity and characterized (Puzon et al., 2002). The reductase activity was NADH- or NADPH-dependent. Reduction of Cr (VI) by H2S produced by the bacterial cells is found in soil environments which are rich in sulfate under anaerobic conditions (Losi et al., 1994). Hydrogen sulfide, produced in acid sulfate soil under reducing conditions, is easily precipitated as FeS in reduced soils (Eary and Rai, 1991) and sediments. Fe (II) and H2S, both microbially produced, are effective reductants of Cr (VI) under reduced conditions as is the FeS (Karnachuk, 1995).
In addition, chromium reducing bacteria also synthesize plant growth promoting substances (Wasi et al., 2008). Therefore, the use of bacteria for reduction/detoxification of chromium is one of the preferred choices and is considered as cost effective approach in bioremediation technologies. The present study was therefore under taken (1) To determine the resistance pattern of soil bacteria to heavy metals and antibiotics and (2) To check chromium reduction under varying pH and chromium concentration.
MATERIALS AND METHODS
Collection of soil sample: The soil samples for the isolation of heavy metal resistance microorganisms were collected from the contaminated soils of Abeokuta, Ogun state, Nigeria.
Isolation of bacteria: Bacteria were isolated from the contaminated soils of Abeokuta on nutrient agar medium by spread plate technique. One gram of soil sample was added to a flask containing 100 mL of normal saline solution and was serially diluted. A 10 μL of each suspension was spread plated on solid nutrient agar. Plates were incubated at 28±2°C for 24 h and the bacterial colonies were then purified and preserved on nutrient agar slants for further experiments.
Evaluation of bacterial strains for metal tolerance: The isolated bacterial strains from the contaminated soil were tested for their sensitivity/resistance to three heavy metals viz., chromium, zinc and copper by agar plate dilution method (Holt et al., 1994) using nutrient agar. The freshly prepared agar plates amended with increasing concentration of chromium (0-700 μg mL-1), zinc (0-700 μg mL-1) and copper (0-700 μg mL-1) were spot inoculated (10 μL) with 108 cells mL-1. Plates were incubated at 28±2°C for 72 h and the highest concentration of heavy metals supporting growth was defined as the Maximum Resistance Level (MRL). Each experiment was replicated three times.
Determination of antibiotic sensitivity: To determine susceptibility to antibiotics, the bacterial strains were tested for their sensitivity to ten antibiotics. The reactions to antibiotics were determined by the disc diffusion method (Bauer et al., 1966). The bacterial strains were grown in nutrient broth at 28±2°C for 24 h. A 0.1 mL of the over-night grown culture was spread on the surface of nutrient agar. The antibiotic discs of known potency were then placed on the agar surface and the plates were incubated at 28±2°C for 24 h. The zones of inhibition around the antibiotic discs were measured and the strains were classified as Resistant (R), Intermediate (I) and Susceptible (S), following the standard antibiotic disc sensitivity testing method (Difco Laboratories, 1984) to the following antibiotics: Septrin (30 μg), Chloramphenicol (30 μg), Ciprofloxacin (10 μg), Sparfloxacin (10 μg), Amoxicillin (30 μg), Augmentin (30 μg), Gentamycin (10 μg), Pefloxacin (30 μg), Streptomycin (30 μg) and Tarivid (10 μg).
In vitro assay of hydrogen cyanide and ammonia: Hydrogen cyanide production by bacterial isolates was detected by the method of Bakker and Schippers (1987). For HCN production, the bacterial strains were grown on an HCN induction medium (30 g tryptic soy broth, 4.4 g glycine and 15 g agar L-1) at 28±2°C for four days. For each bacterial isolate, 100 μL of 108 cells mL-1 was placed in the centre of the petri plates. A disk of Whatman filter paper No. 1 dipped in 0.5% picric acid and 2% Na2CO3 was placed at the lid of the petri plates. Plates were sealed with parafilm. After four days incubation at 28±2°C, an orange brown colour of the paper indicating HCN production was observed.
For ammonia production, the bacterial strains were grown in peptone water (g L-1: peptone 10 g, NaCl 5 g, pH 7) and incubated at 30±2°C for four days. One milliliter of Nessler reagent was added to each tube and the development of yellow color indicating ammonia production was recorded (Dye, 1962).
Chromium (VI) reduction: To assess the effect of pH on hexavalent chromium [Cr (VI)] reduction in vitro, the Nutrient Broth (NB) was amended with 100 μg mL-1 of Cr (VI) and the autoclaved medium was adjusted to pH 5, 6, 7, 8 and 9 with 1M HCl or 1 M NaOH and incubated at 28±2°C for 120 h. Further, to assess the effect of different concentrations (0, 50,100 and 150 μg mL-1) of Cr (VI), the K2Cr2O7 were amended in Nutrient broth and incubated at 28±2°C for 120 h. For Cr (VI) reduction, 1 mL culture from each flask was centrifuged (6000 rpm) for 10 min at 10°C and Cr (VI) in the supernatant was determined by 1, 5-diphenyl carbazide method (Eaton et al., 1992) upto 120 h. Briefly, the test samples were acidified (pH 1-2) and 1, 5 diphenyl carbazide (50 μg mL-1) was added and Cr (VI) concentration was detected by spectrophotometer (spectronic 20D) at 540 nm.
Data of three replicates were subjected to statistical analysis using pair samples T test with significant level of p<0.05. The values indicate the Mean±SD of three replicates.
RESULTS AND DISCUSSION
Evaluation of bacterial for metal tolerance: The bacterial strains were evaluated for their tolerance to various concentrations of chromium (VI) and other metals like zinc and copper using agar plate dilution method (Fig. 1). Generally, bacterial strains showed a varied level of tolerance to heavy metals. Among the bacterial strains, Bacillus spp. PZ3 and Streptococcus spp. PZ4 showed highest tolerance to Chromium (vi) at concentration 700 μg mL-1, Pseudomonas spp. PZ1 and Streptococcus spp. PZ2 showed highest tolerance to Copper and Zinc at concentration 700 and 700 μg mL-1, respectively. There are reports which have shown the resistance of bacteria to heavy metals (Wani and Khan, 2013). There are many reports which have shown varied level of tolerance by bacteria. This varied level of resistance could be due to the variation in growth conditions employed (Rajkumar et al., 2005). For example, Rhizobium leguminosarum have shown a tolerance level of 92.9 μM to zinc (Delorme et al., 2003) while Rhizobium species isolated from nodules of Trifolium repense tolerated 300 mg kg-1 nickel and when grown in nickel amended soil could nodulate the legume crop effectively (Smith and Giller, 1992). In the present study, Bacillus spp. PZ3 and Streptococcus spp. PZ4 showed highest tolerance to Chromium (vi) at a concentration of 700 μg mL-1 to each strain, Pseudomonas spp. PZ1 and Streptococcus spp. PZ2 showed highest tolerance to Copper and Zinc at concentration 700 and 700 μg mL-1, respectively. Bacterial strains showed a high tolerance to chromium which was followed by copper and then zinc. The metal tolerant strains were characterized by physiological, morphological and biochemical characteristics. The strain PZ1 and PZ6 was characterized as Pseudomonas spp. PZ2 and PZ4 was characterized as Streptococcus spp. PZ3 was characterized as Bacillus spp. while PZ5 was characterized as Micrococcus spp.
Rhizobium leguminosarum biovar trifolii isolated from sewage sludge treated soil showed a high tolerance level of 0.24-0.26 mM to Ni2+ and 6.0-8.0 mM to Zn2+ (Purchase and Miles, 2001). Similarly, metal tolerance by Rhizobium, Bradyrhizobium and Azotobacter (Pajuelo et al., 2008) and varying level of resistance among other PGPR (Bacillus and Pseudomonas) have also been reported (Yilmaz, 2003; Thacker et al., 2007; Wani et al., 2008).
Antibiotic resistance of bacterial strains: Resistance to antibiotics varied from one bacterial strain to another strain (Table 1).
| Fig. 1: | Maximum tolerance level shown by different bacterial isolates |
| Table 1: | Resistance pattern of bacterial species to various antibiotics |
| Table 2: | Plant growth promoting activities of metal resistant bacterial isolates |
Among bacterial species, 100% of strains were resistant to Septrin, Chloramphenicol, Sparfloxacin, Amoxacillin, Augumentin, Tarivid and Streptomycin, 83.33% were resistant to Gentamycin and Pefloxacin whereas 33.33% were resistant to Ciprofloxacin. Bacterial resistance to antibiotics is an emerging problem these days. Resistance to antibiotics by microbes could be due to change in the genetic makeup, can be due to a genetic mutation or by transfer of antibiotic resistant genes between organisms in the environment (Spain and Alm, 2003). With these considerations, the antibiotic resistance among PGPR was studied which differed from antibiotic to antibiotic for all the PGPR strains. Multi antibiotic resistances shown by the bacterial strains (e.g., Bacillus spp. PZ3 and Pseudomonas spp. PZ6) may be because of their tolerance to metals which have been reported in many studies (Yilmaz, 2003; Verma et al., 2001). It has been observed that metal and antibiotic resistant organisms can adapt faster to metal stress in the environment due to R-factors than by mutation and natural selection (Silver and Misra, 1988). Similar observations on antibiotics resistance by PGPR strains have been reported (Thacker et al., 2007). The variation in the resistance to many tested antibacterial drugs (antibiotics) may possibly be due to the differences in growth conditions and exposure of PGPR to stress conditions or toxic substance as well as presence or absence of resistance mechanisms that could be encoded either by chromosome and/or R-plasmid (Spain and Alm, 2003).
In vitro assay of HCN and ammonia: The plant growth promoting rhizobacterial strains were tested further for the synthesis of ammonia and hydrogen cyanide using peptone water and HCN induction medium, respectively (Table 2). Generally, all PGPR strains were found positive for ammonia while PZ3, PZ4 were found to be positive for HCN. In the present study bacterial strains were positive to plant growth promoting activities and produced substantial amount of HCN and Ammonia. The ammonia released by these bacterial strains plays a signaling role in the interaction between plant growth promoting bacteria and plants (Becker et al., 2002).
| Fig. 2: | Effect of pH on Cr (VI) reduction ability of Streptococcus spp. PZ4 after 120 h of growth in nutrient broth |
Moreover, the ammonia released by the bacterial strain is known to increase the glutamine synthetase activity (Sood et al., 2002). In addition, ammonia transporters found in several PGPR are thought to be involved in the reabsorption of NH4+ released as a consequence of NH3 diffusion through the bacterial membrane (Van Dommelen et al., 1997).
Chromium (VI) reduction: Chromium is an environmental pollutant released from various industries including tanneries, metal cleaning and processing, chromium plating, wood processing and alloy formation. Chromium [Cr (VI)] is the most toxic and carcinogenic (Kamaludeen et al., 2003) due to its high solubility, rapid permeability and their intracellular proteins and nucleic acids (Reeves et al., 1983). The reduction of Cr (VI) leads to the formation of stable, less soluble and less toxic Cr (III) and is thus, a useful process for remediation of Cr (VI) affected environments (Thacker et al., 2007). Thus detoxifications of chromium by bacterial strains is thus a good technique to clean the environment from chromium. Therefore, the present study was designed to determine the Cr (VI) reducing ability of the metal tolerant strains.
Among all the strains only one strain Streptococcus species PZ4 showed chromium reducing ability under in vitro conditions as this strain was highly resistant to chromium (VI). This study was carried out to access the (1) Effect of different pH values on the reduction of Cr (VI) and (2) The effect of chromate concentration on chromium (VI) reduction.
The effect of different pH values on the reduction of chromium (VI) is shown in Fig. 2. Maximum reduction (85%) of chromium (VI) was observed at pH 7 by Streptococcus spp. PZ4. Similarly, PGPR isolates Streptococcus spp. PZ4 reduced the chromium considerably at pH 5 (51.25), pH 6 (72.5%), pH 8 (67.5%) and at pH 9 (45%), respectively, at a concentration of 100 μg Cr mL-1 after 120 h of incubation.
In this study, the chromium reducing ability of PGPR strain was assessed using nutrient broth supplemented with 50, 100 and 150 μg mL-1 of K2Cr2O7 in order to determine the effect of chromium (VI) reducing ability of the selected culture under in vitro conditions (Fig. 3). The time for total reduction of chromium (VI) increased with increase in the concentration of chromium (VI). During this study, the complete reduction of chromium (VI) occurred after 120 h by Streptococcus spp. PZ4 (Fig. 2) at 50 μg mL-1 of chromium.
| Fig. 3: | Effect of Cr (VI) on Cr (VI) reduction ability of Streptococcus spp. PZ4 in nutrient broth after 120 h of incubation |
The PGPR strain Streptococcus spp. PZ4 reduced chromium (VI) at concentration of 50 μg Cr mL-1 (47.5%), 100 μg Cr mL-1 (91.25%) and 150 μg Cr mL-1 (134.1%), respectively. Our study is in correlation with the study of Yang et al. (2009) who also observed considerable reduction of chromium.
CONCLUSION
This study concludes that the bacterial strains not only tolerated heavy metals, antibiotics, produced plant growth promoting substances but also reduced Chromium (VI) under different pH and chromium concentration. Due to multifarious properties expressed by the bacterial strains, these strains could therefore be used as bioremediators of metals in soils contaminated with heavy metals and can also increase the yield of various crops under heavy metal contamination.