Profiling of heavy metal(loid)-resistant bacterial community structure by metagenomic-DNA fingerprinting using PCR–DGGE for monitoring and bioremediation of contaminated environment
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Frequent exposure of microbes to hazardous metalloids/heavy metals in contaminated environment results in the development of heavy metal(loid)-resistance properties. The study attempted to assess the profile of elevated arsenic (As), cadmium (Cd) and mercury (Hg)—resistant bacterial community structures of sludge (S1, India), sludge and sediment (S2 and S3, Japan) and sediment (S4, Vietnam) samples by metagenomic-DNA fingerprinting using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) for monitoring and bioremediation of hazardous metal(loid) contamination in environment. The results revealed that As-resistant bacteria were dominant compared to Cd- and Hg-resistant bacteria with higher species diversity (Lysinibacillus sp., Uncultured soil bacterium clone, Staphylococcus sciuri, Bacillus fastidiosus, Bacillus niacini, Clostridium sp. and Bacillus sp.) in S1 and S4 than that of S2 and S3 samples. The occurrence of dominant As-resistant bacteria may indicate arsenic contamination in the investigated coastal habitats of India, Japan and Vietnam. The As-, Cd- and Hg-resistant bacteria/bacterial consortiums showed appreciable uptake ability of respective metal(loid) (0.042–0.125 mg As/l, 0.696–0.726 mg Cd/l and 0.34–0.412 mg Hg/l). Therefore, it might be concluded that the profiling of metalloids/heavy metal-resistant bacterial community structure by metagenomic-DNA fingerprinting using PCR–DGGE could be used to explore high metal(loid)-resistant bacteria for applying in metal(loid) bioremediation and as an indicator for monitoring hazardous metal(loid) contamination in environment.
KeywordsMetal(loid) Resistant Metagenomics Bacterial diversity Environmental contamination Bioremediation
Indiscriminate and uncontrolled discharge of hazardous heavy metal(loid) such as arsenic (As), cadmium (Cd) and mercury (Hg) by various anthropogenic as well as geogenic activities contaminates the environment posing severe health hazardous and detrimental impacts on all forms of life (Bidstrup 1964; Gupta and Gupta 1998; Bhakta 2017; Signes-Pastor et al. 2017; Hoover et al. 2017). The contamination of hazardous metalloids/heavy metals impacts on the qualitative and quantitative structures of microbial communities in the environment. Frequent exposure of microbial community to contaminated hazardous metalloids/heavy metals results in the acquisition of heavy metal(loid)-resistance properties by the process of metal(loid) homeostasis, facilitation of heavy metals mobility (Gadd 1990; Bhakta 2016, 2017), alteration of metabolic activity and diversity (Giller et al. 1998). The exploration of potential resistant bacteria from the environment for microbial heavy metal(loid) remediation is an emerging field. The heavy metal-resistant bacteria, Lactobacillus reuteri and Enterococcus faecium have been isolated from coastal sediment samples and characterized for use as hazardous metals removing agents (Bhakta et al. 2012a, b). Similarly, other studies have employed microbial species such as Escherichia coli, Bacillus subtilis, Saccharomyces boulardii, Enterococcus faecium, Staphylococcus aureus and Vibrio fluvialis to remove hazardous pollutants from aquatic environment (Min-sheng et al. 2001; Wei et al. 2009; Figueiredo et al. 2016; Saranya et al. 2017).
The diversified environment, especially the vast richness of soil microbial niches, is the best source of novel microorganisms with novel molecules that can provide various biotechnological applications. It is also obvious that the simple cultivation-dependent and colony screening approaches are unable to identify the majority of microorganisms present in soil, and so the vast amount of potential soil microorganisms remain unidentified., Metagenomics, the analysis of the entire genetic complement of a particular habitat (Handelsman et al. 1998, 2004) by DNA fingerprinting using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE), has emerged as one of the key technologies (Logue et al. 2008; Kumar et al. 2015) that has allowed access to a wide diversity of individual genes and their products as well as analysis of entire operons encoding biosynthetic or degradative pathways (Handelsman 2004; Pettit 2004; Streit and Schmitz 2004; Schmeisser et al. 2007; Dinsdale et al. 2008; Kunin et al. 2008, Umar et al. 2017). As such, the metagenomic-16S rDNA/rRNA fingerprinting technology has extensively expanded our knowledge of microbial diversity and revealed that the range of soil microbial diversity is between 3000 and 11,000 genomes per gram of soil with less than 1% being accessible through cultivation techniques (Torsvik and Ovreas 2002; Curtis and Sloan 2004). Metagenomics also makes it possible to answer key ecological questions by enabling scientists to relate potential functions to specific microorganisms within multispecies soil communities by profiling microbial diversity using DNA fingerprinting (Smalla et al. 2007; Dinsdale et al. 2008; Kunin et al. 2008; Campbell et al. 2009; Martínez-Alonso et al. 2010). Very few studies have been performed on the screening of heavy metal(loid)-resistant bacteria from metagenomic soil samples using PCR–DGGE (Qing et al. 2007; Altimira et al. 2012). Additionally, taking the advantage of metal(loid)-resistant properties, microorganisms are potentially using in metal(loid) removing/uptaking process for the remediation of contaminated environment (water and soil) in recent years (Bhakta et al. 2012a, b; Sinha et al. 2012; Bhakta et al. 2014; Jafari and Cheraghi 2014; Watts et al. 2015; Carpio et al. 2016; Saranya et al. 2017).
The detailed profiling of hazardous metal(loid)-resistant bacteria in environmental samples (soil/sediment/sludge) by metagenomics technology has not been widely studied so far. Therefore, the objectives of the present study have been aimed to profile the elevated As-, Cd- and Hg-resistant bacteria by metagenomic-DNA fingerprinting using PCR–DGGE of sediment and sludge samples for exploration and determination of potential toxic and hazardous metal(loid)-resistant bacteria/bacterial consortium and application of them to remediate these hazardous metal(loid)-contaminated environment as a promising bioremediation technology.
2 Materials and methods
2.1 Study area and sampling
2.2 Metal(loid) solution
The arsenic (As), cadmium (Cd) and mercury (Hg) solutions were prepared from stock solutions of arsenic trioxide (As2O3), cadmium chloride (CdCl2) and mercuric chloride (HgCl2) (Cica-Reagent, Kanto Chemical Co., Inc., Tokyo, Japan), respectively, and sterilized before use.
2.3 Enrichment culture of resistant bacteria/bacterial consortium
2.4 Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE)
One milliliter of preserved enriched cultured broth was centrifuged; supernatant was discarded leaving the metal(loid)-resistant bacterial community remaining. The total DNA of the bacteria was extracted following the methods described by Ruiz-Barba et al. (2005) and was used as a source of DNA template for PCR. The 16S rDNA fragments were amplified by PCR using the universal primers, 341F (5´-CCTACGGGAGGCAGCAG-3´) and 534R (5´-ATTACCGCGGCTGCTGG CA-3´) (Invitrogen) and the thermocycler PC818 (ASTEC program temperature control system). A GC clamp (5′-cgcccgccgcgcgcggcgggcggggcgggggcacgggggg-3′) was linked to the first primer to obtain F341-GC (5′-cgcccgccgcgcgcggcgggcggggcgggggcacggggggCCTACGGGAGGCAGCAG-3′). The PCR system (40 µl) contained 20 µl of AmpliTaq Gold® 360 Master Mix with 1 µl 360 GC Enhance (Applied Biosystems), 4 µl of each primer (341F-GC clamp and 534R), 9 µl nuclease free water and 2 µl template DNA. The thermocycle program was as follows: 95 °C for 10 min; 30 cycles of 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min; and a final extension step at 72 °C for 7 min. The PCR products were detected by electrophoresis on a 1.2% agarose gel, stained with ethidium bromide and visualized under UV light.
The DGGE was performed in a DGGE apparatus (Bio-Rad, Richmond, CA, USA) at 58 °C on 8% polyacrylamide gel with denaturing ranges from 30 to 50%. The electrophoresis running time was 2.5 h at 150 V. The gel (16 × 16 cm) was stained by SYBR Gold; bands were visualized using a UV transilluminator and photographed.
2.5 Identification of resistant bacterial strain
The DNA containing bands from the polyacrylamide gel were purified by polyacrylamide gel extraction kit (QIAEX® II, QIAGEN). The sequencing of purified DNA was performed using an automated DNA sequencer (Applied Biosystems, 3100-Avant Genetic Analyzer) (Bhakta et al. 2012b, 2014). This sequence was used for bacterial identification using BLAST (Basic logical alignment search tool) at NCBI and DDBJ.
2.6 Heavy metal(loid) bioremediation of resistant bacteria/bacterial consortium
The preserved resistant bacteria/bacterial consortium was cultured in TSB for 24 h at 37 °C to obtain fresh culture. Bacterial growth was not found in inoculums of the S4 and S1 samples of the Cd- and Hg-supplemented TSB, respectively. The bioremediation study was executed by measuring the heavy metal(loid) uptake of the resistant bacteria/bacterial consortium cells using the method described by Bhakta et al. (2012b). Freshly (24 h) cultured bacterial cells were harvested, centrifuged to pellet the cells and washed by sterilized milli-Q water thrice. The cells (40 mg [wet weight]) were resuspended in 10 ml of sterile 1 mg/l As, Cd or Hg solution, in triplicate. After incubation at 37 °C, samples were collected at 24 and 48 h, centrifuged to pellet cells and the supernatant was passed through a 0.25 μm filter (Advantec, Tokyo) for analysis.
The As, Cd and Hg content of the samples were analyzed using an ICP–AES (ICPS-1000IV; Shimadzu, Tokyo), an atomic absorption spectrophotometer (AA-6800; Shimadzu, Tokyo) and a RA-3 Mercury Analyzer (Nippon Instruments Corporation, Tokyo), respectively (Bhakta and Munekage 2008, 2010; Bhakta et al. 2012a, b, 2014).
3 Results and discussion
3.1 Enrichment culture of resistant bacteria/bacterial consortium
As, Cd and Hg content in the sludge/sediment samples employed in study
1938 ± 20
283 ± 11
1016 ± 35
1860 ± 10
1407 ± 12
530 ± 19
813 ± 16
108 ± 25
23 ± 3.5
207 ± 15
178 ± 22
114 ± 16
Bacterial growth from all samples was clearly observed in the TSB media provided with As (100 mg/l) except sample S2, whereas clear growth of bacteria was not visible in all samples cultured in TSB media supplemented with Cd (100 mg/l) and Hg (20 mg/l). These results suggest that S1, S3 and S4 samples contain a high number and diversity of As-resistant bacteria, whereas very few bacteria were resistant to 100 mg/l Cd and 20 mg/l Hg concentrations in the four samples. It can be conferred herein that majority microbes of employed samples were commonly high resistant to As compared to that of Cd and Hg, since the toxic impacts of Cd and Hg against microbes are probably higher than that of As, which strongly inhibit to develop the resistant properties in most of the microbes. Additionally, it is known that some species of autotrophic and heterotrophic microorganisms use arsenic oxyanions for their regeneration of energy and use arsenate as their nutrient in respiratory process (Lim et al. 2014). Therefore, number and diversity of As-resistant bacteria were higher compared to Cd- and Hg-resistant bacteria.
3.2 Analysis of PCR–DGGE
The results of PCR–DGGE analysis showed that eight resistant bacterial strains A, B, C, D, E, F, G and H were found in samples S1, S2, S3 and S4. Of the eight, 7 types of bacterial strains were appeared as As-resistant. The As-resistant strain A was found in all samples and F appeared in S1, S3, S4 samples, whereas strains C, D and G were exclusively found in S1 and strains E and H were exclusive in sample S4. Only one Cd-resistant strain B was detected in S1, S2 and S3 samples. The strains B and G were appeared as Hg-resistant in S2, S3 and S4 samples. These results indicated that the diversity of As-resistant bacterial species was significantly greater compared to the Cd- and Hg-resistant bacterial species diversity in the four samples. Generally, heavy metal(loid) contamination of the environment is greatly responsible for acquiring the resistance properties of bacteria in the sample investigated. Considering this fact, therefore, the presence of dominant As-resistant bacteria indicates arsenic contamination in the investigated areas of India, coastal area of Japan, and Vietnam, whereas the sludge collected from area S2 in Japan may not be As contaminated due to no apparent As-resistant bacterial dominance and diversity. The bacterial community studied by DGGE revealed that heavy metal contamination in marine sediments changes the bacterial community structure (Yao et al. 2017) and in agricultural soils close to copper and zinc smelters may provoke changes in the composition of soil bacterial community and a decrease of the bacterial diversity (Li et al. 2006; Wang et al. 2007; Altimira et al. 2012). However, changes in the soil bacterial community exposed to heavy metals may vary depending of soil properties, heavy metal bioavailability and the indigenous microbial groups in soil (Ranjard et al. 2006). Bhakta et al. (2012a, b) postulated that microbial communities of coastal sediments and sewage sludge samples acquire resistance properties due to frequent exposure to various heavy metals transported by runoff water. The PCR–DGGE also revealed that the strain B is resistant to both Cd and Hg, while strain G is resistant to both As and Hg, whereas all remaining strains are resistant to only As. This indicated that the strain B and G have acquired multi heavy metal(loid)-resistance ability. Bhakta et al. (2012b) and Qing et al. (2007) also showed high multi-resistance ability of bacteria.
Irrespective of heavy metal(loid) specificity, S1, S2, S3 and S4 samples collectively contain six (A, B, C, D, F and G), three (A, B and G), four (A. B, F and G) and six (A, B, E, F, G and H) bacterial strains, respectively. This clearly suggested that S1 and S4 samples have higher bacterial diversity than the S2 and S3 samples. The high dominance and diversity of bacterial species in the canal sludge of India and coastal sediment of Vietnam samples may be due to the effect of the tropical zones. Bhakta et al. (2012b) revealed that coastal sediments and sewage sludge are the rich source of heavy metal(loid)-resistant bacteria.
3.3 Identification of resistant strain
The BLAST homology search of amplified 16S rDNA nucleotide sequence from the resistant strains revealed similarity to Lysinibacillus sp. (A), uncultured Lactobacillaceae bacterium (B), an uncultured soil bacterium clone (C), Staphylococcus sciuri (D), Bacillus fastidiosus (E), Bacillus niacin (F), Clostridium sp. (G) and Bacillus sp. (H). Bacillus cereus and Enterobacter cloacae were previously identified as Cd-resistant bacterial strains from soil of Pb–Zn tailing in a suburb of Beijing City (Quing et al. 2007). A number various heavy metal(loid)-resistant Lactobacillus sp., Bacillus sp. and Enterococcus sp. have also been isolated from coastal sediments and sludge samples of India, Japan and Vietnam (Bhakta et al. 2012a, b), Enterobacter sp. and Klebsiella pneumoniae from wastewater (Abbas et al. 2014), and Vibrio fluvialis from industrial effluents (Saranya et al. 2017). Figueiredo et al. (2016) identified aerobic Hg-resistant in the Tagus Estuary (Portugal) for Hg bioremediation.
3.4 Heavy metal(loid) bioremediation of resistant bacteria/bacterial consortium
In case of As, As-resistant bacteria/bacterial consortium of S1 removed higher percentage (90–190%) of As compared to that of the As-resistant bacteria/bacterial consortiums of S2, S3 and S4, which might be due to the presence of the high dominance and diversity of As-resistant bacterial species and especially for the presence of uncultured soil bacterium clone (C) in S1 as proved from the enrichment culture and PCR–DGGE analysis. There was no remarkable Cd or Hg removal difference among S1, S2 and S3 of Cd or among S2, S3 and S4 of Hg-resistant bacteria/bacterial consortiums obtained from samples. Since only uncultured Lactobacillaceae was found as Cd-resistant bacterium in S1, S2 and S3 samples, which showed almost similar Cd removal rate in Cd bioremediation study, and in Hg removal study, almost similar amount of Hg removal was estimated in S2, S3 and S4 samples by uncultured Lactobacillaceae bacterium and Clostridium sp. constituting Hg-resistant bacterial consortium. The heavy metal(loid) (As, Cd and Hg) removal performances of corresponding metal(loid)-resistant bacteria/bacterial consortium were temporally increased (Fig. 4). The above results also imply that the resistant bacteria/bacterial consortium can survive in high heavy metal(loid) contaminated environment and can remove appreciable amounts of heavy metal(loid) from ambience probably due to having higher uptake and resistance capacity, which might play a pivotal role in bioremediation process of metalloid and/or heavy metal in environment (Mejias Carpio et al. 2016; Kvasnova et al. 2017). The metal(loid)-resistant bacteria, in particular a mixed consortia are therefore a promising tool to remove metals from an aqueous phase (Bhakta et al. 2012a, b, 2014; Carpio et al. 2014; O’Brien and Buckling 2015; Abbas et al. 2015; Mejias Carpio et al. 2016; Kvasnova et al. 2017). Indeed, Singh et al. (2012) proposed mixed bacterial consortia as an emerging tool to remove hazardous trace metals.
This study has drawn the following conclusions of As-, Cd- and Hg-resistant bacterial community structure of the investigated habitats: (1) the dominance and diversities of As-resistant bacterial species were higher in the investigated samples compared to Cd- and Hg-resistant bacterial species diversities and (2) the dominance and diversities of As-resistant bacterial species in the canal sludge of India and coastal sediment of Vietnam were greater than the remaining samples. The dominant As-resistant bacteria, therefore, indicated arsenic contamination in the investigated habitat of India, coastal habitat of Japan and Vietnam which is also proved by high As content in samples. The metal(loid) uptake properties of the identified resistant bacteria/bacterial consortia demonstrated their potential application for heavy metal(loid) bioremediation. It can be concluded that the profiling of heavy metal(loid)-resistant bacterial community structure of a habitat by metagenomic-DNA fingerprinting using PCR–DGGE is an excellent tool to explore novel heavy metal(loid)-resistant bacteria/bacterial consortia for applying in metal(loid) bioremediation and as an alternative indicator for monitoring and identifying heavy metal(loid) contamination in the environment (water and soil).
Authors are grateful to Japan Society for the promotion of Science (JSPS) for sponsoring research fund and fellowship (FY2009 JSPS postdoctoral fellowship) to Dr. Bhakta to carry out the study. Authors are also especially grateful to Dr. J. K. Pittman for reviewing the manuscript.
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no conflict of interest.
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