Environ Sci Pollut Res DOI 10.1007/s11356-014-2951-3
RESEARCH ARTICLE
Biosequestration of lead using Bacillus strains isolated from seleniferous soils and sediments of Punjab Saurabh Gupta & Richa Goyal & Nagaraja Tejo Prakash
Received: 19 December 2013 / Accepted: 21 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The present study was conducted to isolate and explore bacterial strains with a potential to sequester lead (Pb) and tolerate other heavy metals from industrial effluents and sediments. Out of the six bacterial strains isolated from seleniferous sites of Punjab, three isolates (RS-1, RS-2, and RS-3) were screened out for further growth-associated lead sequestration and molecular characterization on the basis of their tolerance toward lead and other heavy metals. Biomass and cell-free supernatant were analyzed for lead contents using ICP-MS after growth-associated lead sequestration studies in tryptone soya broth (pH=7.2±0.2) under aerobic conditions at 37 °C temperature. Almost 82 % and 70 % divalent lead was sequestered in cell pellets of RS-1 and RS-3, respectively while only 45 % of lead was found in cell pellet of RS-2 in the first 24 h. However, significant biosequestration of lead was observed in RS-2 after 48 h of incubation with concomitant increase in biomass. Simultaneously, morphological, biochemical, and physiological characterization of selected strains was carried out. 16S rRNA gene sequence of these isolates revealed their phylogenetic relationship with class Bacillaceae, a low G + C firmicutes showing 98 % homology with Bacillus sp.
Responsible editor: Robert Duran S. Gupta (*) Department of Microbiology, Mata Gujri College, Fatehgarh Sahib 140406, Punjab, India e-mail: [email protected] R. Goyal Department of Microbiology, Dolphin PG College of Life Sciences, Chunni Kalan 140307, Punjab, India N. T. Prakash Department of Biotechnology & Environmental Sciences, Thapar University, Patiala 147004, Punjab, India
Keywords Metal sequestration . Biotransformation . Bioaccumulation . Lead
Introduction Heavy metals, metalloids, and radionuclides released from ores and industrial activities have become a major concern in terms of environmental pollution (Schaller et al. 2010). Accumulation of these metals/metalloids in different ecosystems leads to an enhanced risk to abiotic and biotic components of associated ecosystem (Luoma and Rainbow 2008). Leaching of these elements and their compounds into soil and ground water facilitate their easy uptake in plants followed by animals through food chains and imparts adverse effects on human health (Nordberg 2003). Even at low concentrations, heavy metals like mercury (Hg), cadmium (Cd), and lead (Pb) directly cause oxidative stress, lipid peroxidation, carcinogenesis, mutagenesis, and neurotoxicity in humans, animals, and plants (Ahmad et al. 2012; Gupta and Ali 2004; Howlett and Avery 1997; Jaroslawiecka and Piotrowska-Seget 2014). Lead is one of the most toxic pollutants in the environment absorbed by plants without any known biological function (Peralta-Videa et al. 2009). Elevated concentration of lead released into an ecosystem through smelting, mining, electroplating, and paint industry become a serious pervasive pollutant. Finally, this is transported to the biological system through ground water followed by its biomagnifications in living beings through the food chain (McLaughlin et al. 1999). Human exposure to higher concentrations of lead (Pb) may lead to renal tubular dysfunction along with proteinuria, glucosuria, and tubular necrosis (Alfven et al. 2002). Inhalation of lead results in metal fume fever with chemical pneumonitis, pulmonary edema, and death (Hayes 2007). The use of biological systems, especially plants and microorganisms, for removal of heavy metals from contaminated
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sites is a fast growing and promising technology with several advantages over traditional methods such as precipitation, filtration, ion exchange, oxidation reduction, electrochemical recovery, membrane separation, and other techniques (Dhankher et al. 2002). Microorganisms use various resistance mechanisms such as permeability barriers, intra- and extracellular sequestration, efflux pumps, enzymatic detoxification, and reduction to overcome heavy metal stress (Nies 1999). Numerous studies on biosorption of lead using different biological material have been reported in recent years. Different living systems such as marine algae, fungi, and yeasts possess significant metal-binding capacity for a wide range of heavy metals and metalloids (Al-Garni 2005; Deng et al. 2007; Halttunen 2007; Kogej et al. 2010; Vieira et al. 2007). In most of the studies dealing with the bio-removal of metals, either dead or resting biomass has been used (Deng et al. 2007; Gadd 1990). Bio sequestration is one of the bioremediation approach where application of a judicious consortium of growing metal-resistant cells ensure better removal through a continuous metabolic uptake of metals after physical adsorption. Bio sequestration may lead to the simultaneous removal of toxic metals, organic loads, and other inorganic impurities, as well as allow optimization through the development of resistant species (Malik 2004). However, there are limited reports on the Pb-sequestration using viable and aerobically growing bacteria. In the present study, three aerobic strains were isolated from selenium-contaminated sites with multiple metal tolerance along with lead to remediate lead contaminated sites. Besides India, selenium contaminated soils have been reported in China, South Africa, Columbia, Argentina, Venezuela, Spain, Bulgaria, Ireland, Algeria, Morocco, Australia, and New Zealand and in some drier regions of the former Soviet Union (Gupta et al. 2010). Present study reports one of the few observations on lead sequestration up to 50 mg/l with actively growing Bacillus sp.
Materials and methods Collection of samples Soil and sludge samples were collected from the Jainpur village of Hoshiarpur–Nawanshahr regions of Punjab in India (75° 55 E; 31° 56 N). Elevated selenium contents in low-lying areas of this region were reported where selenium oxyanions are transported by rain water through seasonal rivulets from the nearby hills of the Shivalik range and deposited in the low-lying areas. The toxic sites are located at the dead ends of the seasonal rivulets. Indigenous microbial communities undergo various self-recovery processes in these polluted sites. Microorganisms have evolved a variety of mechanisms to detoxify these heavy metals, and some even use them for respiration. Hence 1 kg of soil was collected two
times around the year in the months of March and October from these selected patches in sterile polythene bags from a depth of 0–15 cm. Samples were brought to laboratory and were used as such without any prior treatment for enrichment and isolation of lead tolerant bacteria. Five grams of sample was suspended in tryptone soya broth (TSB), pH=7.2±0.2, containing (per liter): 17-g peptone from casein, 3.0-g peptone from soy meal, 2.5-g glucose, 5.0-g NaCl, and 2.5-g K2HPO4 supplemented with lead as lead nitrate (lead as 5.0 mg/l a.i.). Flasks were incubated at 37 °C on an incubator shaker (120 rpm) until growth appeared. A small aliquot was then transferred to the fresh media with increased metal ion concentrations up to 10 mg/l for enrichment of lead tolerant bacteria. After sufficient sub-culturings, a loopful of culture was streaked on tryptone soya agar (TSA) plates to obtain axenic cultures. Plates were incubated under aerobic conditions at 37 °C for 24 h. Colonies with different morphology were picked in pure form and preserved on TSA plates at 4 °C for further studies. Multiple metal tolerance studies These bacterial isolates were further screened for their tolerance and resistance toward different metals. Axenic culture was inoculated in TSB (pH=7.2±0.2) supplemented with different concentrations (i.e., 5.0, 10.0, 25.0, and 50.0 mg/l as an active ingredient) of lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni), arsenic (As), tin (Sn), selenium (Se), zinc (Zn), chromium (Cr), and copper (Cu). Inoculated flasks containing media were incubated at 37 °C for 48 h in shaking conditions (120 rpm) and were observed for appearance of growth. Pb-impacted growth and sequestration studies Three bacterial isolates, namely RS-1, RS-2, and RS-3, were further explored to examine lead (Pb) stressed growth and sequestration studies. An aliquot of these isolates was inoculated in 100-ml TSB (pH =7.2 ± 0.2) supplemented with 50 mg/l lead as PbNO3 in the culture medium and incubated on an orbital shaker at 120 rpm and 37 °C. Simultaneously, a control lacking lead was also maintained for comparison. Initially, growth was monitored after regular interval of 6 h up to 24 h followed by measurement of growth after every 24 h up to 96 h. To check the metal sequestration in the biomass, an aliquot was withdrawn after regular interval of 24 till 96 h. These samples were centrifuged (8,000 rpm at 4 °C for 10 min) for separation of biomass and cell-free supernatant. Biomass and cell-free supernatants were acid digested as described elsewhere (Gupta et al. 2012). In short, biomass and supernatant were acid digested with pre-mixed concentrated nitric acid (HNO3) and perchloric acid (HClO4) in 3:1 ratio. Acid-digested samples
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were diluted with 0.2 % HNO3 and were subjected to inductively coupled plasma mass spectrometry (ICP-MS) analysis to determine distribution of lead in different fractions.
ICP-MS analysis ICP-MS analysis was carried out on PerkinElmer Sciex ELAN DRC-e (Axial Field Technology). The system was operated under inert conditions maintained with argon gas at the flow rate of 15 l through plasma, at 1.2 l and 0.9 l from auxiliary and nebulizer, respectively, throughout the measurement. Pressure was maintained between 60–70 psi. Plasma formation was carried out at 6,000 °C temperature which causes the sample to separate into individual atoms (atomization) by ionization with plasma and detection by mass spectrometer.
Characterization of the bacterial isolates Three selected strains RS-1, RS-2, and RS-3, were characterized in terms of their morphological and biochemical characteristics along with identification and phylogenetic relatedness with other organisms. Biochemical characterization of these strains was carried out at the Microbial Type Culture Collection (MTCC-IMTECH), Chandigarh, India. Molecular characterization was carried out by isolating genomic DNA using standard protocols (Sambrook et al. 1989), amplifying and sequencing the 16S ribosomal RNA (rRNA) gene. PCR amplification was carried out using 27 F and 1492R universal primers (Singh et al. 2010). PCR product was eluted, purified, and sequenced at DNA sequencing facility, Delhi University, Delhi. Sequences were analyzed by using CHECKCHIMERA program of the Ribosomal Database Project (RDP)-II (Maidak et al. 2001). 16S rRNA gene sequences of isolates were compared with those available in GenBank/ EMBL databases using the BLAST program (Altschul et al. 1997). The sequences of closely related strains and uncultured bacteria were retrieved from RDP-II and aligned using multiple alignments CLUSTAL-W program (Thomson et al. 1994; Cole et al. 2007). Phylogenetic dendrograms were constructed by neighbor-joining method in which associated taxa were clustered together by the bootstrap test using MEGA 6 package (Felsenstein 1985; Tamura et al. 2013).
Statistical analysis Metal tolerance, growth, and sequestration experiments were carried out in triplicate and results are presented as mean values along with standard error in the respective figures.
Results Dynamic genetic and biochemical capacity of microorganisms, especially bacteria, has introduced these organisms as an effective tool toward bioremediation of organic and inorganic pollutants such as heavy metals (Roane et al. 2001; Jaroslawiecka and Piotrowska-Seget 2014). In 2004, Malik reviewed the use of growing microorganisms for metal removal and development of technically and economically viable technologies for the treatment of metal-rich effluents. In this work, we have investigated potential of selected bacterial strains toward sequestration of lead for the restoration of naturally and anthropogenic heavy metals-contaminated sites which in turn will limit the transport of these heavy metals in the biological systems. As a part of this study, six lead tolerant bacterial strains were isolated from the selenium-contaminated soils and selected strains were explored for growth-associated lead sequestration along with tolerance of all the isolates for other heavy metals.
Multiple metal tolerance studies A wide variation in minimum inhibitory concentration (MIC) of different metals was observed for different isolates. Isolates RS-1, RS-2, RS-3, and RS-5 were invariably resistant to lead, nickel, and selenium up to 50 mg/l. While minimum inhibitory concentration (MIC) of other metals showed an intermittent range for these isolates (Table 1). RS-4 and RS-6 showed least tolerance for most of the metals with maximum resistance for selenium (50 mg/l) only. RS-5 has already been reported for mercury sequestration (Gupta et al. 2012) while RS-1, RS-2, and RS-3 were further explored in the present study for sequestration of lead.
Table 1 Heavy metal tolerance/MIC (mg/l) profile of different isolates Metals
Lead (Pb) Mercury (Hg) Cadmium (Cd) Nickel (Ni) Arsenic (As) Tin (Sn) Selenium (Se) Zinc (Zn) Chromium (Cr) Copper (Cu)
Isolates RS-1
RS-2
RS-3
RS-4
RS-5
RS-6
50 25 25 50 10 0 50 10 25 10
50 10 50 50 10 0 50 10 25 10
50 25 25 50 10 0 50 25 25 10
25 25 10 10 0 0 50 10 10 5
50 50 25 50 25 0 50 50 50 50
10 0 0 10 0 0 50 10 10 5
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Growth-associated Pb sequestration studies Effect of lead was studied on selected bacterial strains (RS-1, RS-2, and RS-3) in terms of their growth in leadsupplemented culture medium. Growth of these isolates was monitored as increase in optical density over a period of 96 h with concomitant increase in biomass (Fig. 1). The isolates showed typical growth pattern in both control (without lead) and lead (Pb)−containing media. An insignificant lag phase has been observed in RS-1 and RS-3 (Fig. 1a, c) with little effect of lead on starter culture. Besides this, limited effect of lead was observed in subsequent growth phases of RS-1 and RS-3 as indicated by comparable growth in both control and lead supplemented media during stationary phase (Fig. 1a, c). In RS-2, comparatively longer generation time was observed in lead-supplemented media as compared to all other isolates as indicated by optical density measurement (Fig. 1b). Similar slow growth rate was also observed in control of RS-2 with respect to other isolates (Fig. 1). In metal sequestration studies, profound sequestration of metal was observed in biomass of RS-1 followed by RS-3 during the first 24 h as indicated by amount of lead quantified in the cell pellet as compared to the cell-free supernatant (Fig. 2a, c). Approximately, 82 % of metal accumulation was observed in RS-1 followed by 70 % metal sequestration in RS-3. RS-2 showed comparatively less metal sequestration during the first 24 h of incubation (Fig. 2b). Only 45 % of metal was accumulated in the biomass that corroborates with the relatively slow growth of RS-2. Even after prolonged incubation of RS-1 and RS-3 up to 96 h, there was no further significant increase in sequestration of lead observed. On the contrary, substantial increase in biosequestration of lead was observed in RS-2 up to 48 h. After 96 h of incubation, a slight increase of lead was observed in cell-free supernatant of all the isolates (Fig. 2). Characterization and identification of isolates These bacterial strains (RS-1, RS-2, and RS-3) were characterized in terms of their morphological, physiological, and biochemical characteristics. All the isolates were invariably rod shaped and stained Gram-positive, were motile, spore
forming, and did not produce any pigment. Physiologically, RS-3 tolerated a wide range of pH (5.0–11.0), temperature (15–45 °C) and a high salt concentration up to 10 %w/v followed by an intermediate range with RS-1 and a narrow range by RS-2 (Table 2). Out of these three isolates, RS-1 and RS-3 were found catalase and oxidase positive with no nitrate reduction while RS-2 exceptionally reduced nitrate and was oxidase negative. Analysis of the 16S rRNA gene sequence by multiple sequence alignment (BLAST) indicated 99 % homology of all the three strains to Bacillus sp. with 98 % alignment coverage over ~1.5 kb. The sequence was assigned the GenBank accession number HM179550, HM179551, and HM179552. Classification of these isolates to genera Bacillus was confirmed using the RDP-II Classifier (Wang et al. 2007) and evolutionary history was inferred using the maximum composite likelihood method (Fig. 3).
Discussion Remediation of heavy metals-contaminated sites through common physico-chemical techniques is expensive and not eco-friendly. Hence, the use of biotechnological approaches such as use of different microorganisms has received an immense attention as alternative tool (Iyer et al. 2005). A lot of work has been published about the genetic and biochemical mechanisms that microorganisms use to withstand metal toxicity with descriptions of how microorganisms physiologically adapt to metal stress (Chen et al. 2013; Sulaymon et al. 2013; Jaroslawiecka and Piotrowska-Seget 2014). No single strategy has been adopted by microorganisms for survival under heavy metals-stressed conditions. A number of diverse and complex mechanisms of resistance and adaptation such as reduced uptake of toxic metal, efflux, sequestration, metabolic bypass, and chemical modifications have been reported (Gonzalez et al. 2010; Lemire et al. 2013; Rico et al. 2013). Biosorption is a process in which dead and inactive biomass retain relatively high quantities of heavy metals including lead by passive sorption and/or complexation (Veglio and Beolchini 1997; Yetis et al. 2000; Deng et al. 2007). However, the low-binding capacity of biomass for certain recalcitrant metals and failure to effectively remove metals
Fig. 1 Comparative growth kinetics of bacterial strains with and without Pb. 1a RS-1; 1b RS-2; 1c RS-3
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Fig. 2 Comparative bioaccumulation of Pb in different bacterial strains. 2a RS-1; 2b RS-2; 2c RS-3
Table 2 Morphological, physiological, and biochemical characteristics of the RS-1, RS-2, and RS-3
Characters Morphological Cell shape Gram stain Motility Capsule Colony morphology Pigmentation Physiological Growth at 4 °C Growth at 42 °C Growth at 45 °C Growth with 1 % NaCl Growth with 5 % NaCl Growth with 7 % NaCl Growth with 10 % NaCl Growth at pH 2 Growth at pH 5 Growth at pH 8 Growth at pH 11 Biochemical characteristics Indole utilization Methyl red Voges Proskeur Citrate utilization Nitrate reduction H2S production Urease Starch hydrolysis Casein hydrolysis Lipid hydrolysis Gelatin liquefaction
NA nutrient agar
Oxidase Catalase Carbohydrate fermentation test Sucrose Glucose Lactose
RS-1
RS-2
RS-3
Rod shape Gram + ve + − Circular, flat, undulate margins −
Rod shape Gram + ve + − Large, flat, opaque, irregular, undulate margins −
Rod shape Gram variable + + Smooth, undulate margin Yellow/ white
− − − + +
+ − − + +
− + + + +
− − − − + +
− − − + + −
+ + − + + +
− + + − −
− − + + +
− + + + −
− − + + + +
− − + + + +
− − − + + +
+ +
− +
+ +
+ − +
− + −
+ + ±
Environ Sci Pollut Res Fig. 3 Evolutionary relationships of RS-1, RS-2, and RS-3 with other taxa using the neighborjoining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method
from real industrial effluents limit this approach. Hence, the use of actively growing metal-resistant cells ensures better removal through a combination of bioprecipitation, biosorption, and continuous metabolic uptake of metals after physical adsorption (Malik 2004; Volesky 1990). Previously, a number of bacteria have been reported for uptake and transformation of heavy metals and metalloids such as mercury, cadmium, and selenium (Gupta and Jashan 2014; Kumar et al. 2012; Dhanjal and Cameotra 2010). Similarly, in the present study, growth-associated uptake of lead in the biomass has been reported. This observation was in corroboration with growth of the cultures. Growth-associated metal tolerance has been recently reported in Pseudomonas aeruginosa with no data on bioaccumulation capacity
(Mohamed and Abo-Amer 2012). Certain other bacteria such as Alcaligenes faecalis, P. aeruginosa, and B. iodinium were reported with a potential to remove Pb in growth-associated metal detoxification studies (Jaysankar et al. 2008). Growth and metabolism-dependent metal removal involve metal precipitation as sulfides, complexation by siderophores and other metabolites, sequestration by metal-binding proteins and peptides, transport and intracellular compartmentation (White et al. 1995). Bacillus sp. has been observed to form surface layer (S-layer) protein that can be used as a template for sequestration of heavy metals from the industrial effluent (Merroun 2007). Extracellular polymeric substances (EPS), secreted by bacteria, results in the formation of biofilms, which facilitates in situ metal sequestration under pilot scale
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(Edwards and Kjellerup 2013). Exopolysaccharides secreted by Halomonas sp. have net negative charge due to high uronic acids content and play a vital role in sequestration of heavy metals (Gutierrez et al. 2012). In this study, sequestration of lead was carried out in complex medium with high content of dissolved organic carbon (DOC) which might have played an important role in the sequestration of lead. Initial slow growth of RS-2 with concomitant less sequestration of lead may be attributed to inherited property of this bacterial strain toward DOC present in medium. While speciation of intracellular lead was not performed in the present study, lead uptake and transformation was quite evident. Formation of lead sulfide was speculated from change in biomass color from cream to black (data not shown). Over 96 h, the profile of total lead in various fractions viz., cell-free supernatant and biomass indicated that the organism diverts further lead uptake by its expulsion in the supernatant (may be immobilized form) although the expelled fraction is always much less than the accumulated fraction. Since all the isolates grow well in significantly high concentrations of lead and other heavy metals with the potential to sequester lead inside the biomass, these findings indicate efficient detoxification and transformation mechanisms harnessed by these organisms for high concentration of lead metal/cation.
Conclusion This study thus presents salient findings on growth-associated lead sequestration by Bacillus strains isolated from seleniferous sites of Punjab. Exploitation of these strains toward the accumulation of heavy metals within the biomass may provide a useful approach for remediating lead contamination present in various industrial effluents. Interestingly, this is the first report on viable and actively growing Bacillus strains capable of Pb ion sequestration.
References Ahmad E, Zaidi A, Khan MS, Oves M (2012) Heavy metal toxicity to symbiotic nitrogen-fixing microorganism and host legumes. In: Zaidi A, Wani PA, Khan MS (eds) Toxicity of Heavy Metals to Legumes and Bioremediation. Springer Verlag Wein, New York, pp 29–44 Alfven T, Jarup L, Elinder CG (2002) Cadmium and lead in blood in relation to low bone mineral density and tubular proteinuria. Environ Health Perspect 110:699–702 Al-Garni SM (2005) Biosorption of lead by Gram-negative capsulated and non-capsulated bacteria. Water SA 31:3 Altschul SF, Thomas LM, Alejandro AS, Zhang J, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acid Res 25:3389–3402
Chen J, Qin J, Zhu Y, Lorenzo V, Rosen BP (2013) Engineering the soil bacterium Pseudomonas putida for arsenic methylation. Appl Environ Microbiol 79:4493–4495 Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohidden AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tieje JM (2007) The ribosomal database project (RDP-II): introducing my RDP space and quality controlled public data. Nucl Acid Res 35(Database issue):D169–D172 Deng L, Su Y, Su H, Wang X, Zhu X (2007) Sorption and desorption of lead (II) from wastewater by green algae Cladophora fascicularis. J Haz Mat 143:220–225 Dhanjal S, Cameotra SS (2010) Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil. Microb Cell Fact 9:52 Dhankher OP, Li Y, Rosen BP, Shi J, Salt D, Senecoff JF, Sashti NA, Meagher RB (2002) Engineering tolerance and hyper accumulation of arsenic in plants by combining arsenate reductase and gammaglutamylcysteine synthetase expression. Nat Biotechnol 20:1140– 1145 Edwards S, Kjellerup BV (2013) Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals. Appl Microbiol Biotechnol 97:9909–9921 Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791 Gadd GM (1990) Heavy metal accumulation by bacteria and other microorganisms. Experientia 46:834–840 Gonzalez AG, Shirokova LS, Pokrovsky OS, Emnova EE, Martinez RE, Santana-Casiano JM, Gonzalez-Davila M, Pokrovski GS (2010) Adsorption of copper on Pseudomonas aureofaciens : Protective role of surface exopolysaccharides. J Coll Inter Sci 350:305–314 Gupta VK, Ali I (2004) Removal of lead and chromium from waste-water using bagasse fly ash—a sugar industry waste. J Colloid Interface Sci 271:321–328 Gupta S, Nirwan J (2014) Evaluation of mercury biotransformation by heavy metal-tolerant Alcaligenes strain isolated from industrial sludge. Int J Environ Sci Technol. doi:10.1007/s13762-013-0484-9 Gupta S, Prakash R, Tejoprakash N et al (2010) Selenium mobilization of Pseudomonas aeruginosa (SNT-SG1) isolated from seleniferous soils from India. Geomicrobiol J 27:35–42 Gupta S, Goyal R, Nirwan J, Cameotra SS, Tejoprakash N (2012) Bio sequestration, transformation and volatilization of mercury by Lysinibacillus fusiformis isolated from industrial effluent. J Microbiol Biotechnol 22:684–689 Gutierrez T, Biller DV, Shimmield T, Green DH (2012) Metal binding properties of the EPS produced by Halomonas sp. TG39 and its potential in enhancing trace element bioavailability to eukaryotic phytoplankton. Biometals 25:1185–1194 Halttunen T (2007) Removal of lead, cadmium and arsenic from water by lactic acid bacteria. Dissertation, Department of Biochemistry and Food Chemistry, University of Turku, Turku Hayes AW (2007) Principles and methods of toxicology. Taylor & Francis press, Philadelphia Howlett NG, Avery SV (1997) Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation. Appl Environ Microbiol 63:2971–2976 Iyer A, Mody K, Jha B (2005) Biosorption of heavy metals by a marine bacterium. Mar Poll Bull 50:340–343 Jaroslawiecka A, Piotrowska-Seget Z (2014) Lead resistance in micro-organisms. Microbiology 160:12–25 Jaysankar D, Ramaiah N, Vardanyan L (2008) Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar Biotechnol 10:471–477 Kogej A, Likozar B, Pavko A (2010) Lead Biosorption by selfimmobilized Rhizopus nigricans pellets in a laboratory scale packed
Environ Sci Pollut Res bed column: mathematical model and experiment. Food Technol Biotechnol 48:344–351 Kumar A, Gupta S, Cameotra S (2012) Screening and characterization of potential cadmium biosorbent Alcaligenes strain from Industrial effluent. J Basic Microbiol 52:160–166 Lemire JA, Harrison JJ, Turner RJ (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Microbiol 11:371–384 Luoma SN, Rainbow PS (2008) Metal contamination in aquatic environments: science and lateral management. Cambridge University Press, Cambridge Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM (2001) The RDP-II (ribosomal database project). Nucl Acid Res 29:173–174 Malik A (2004) Metal bioremediation through growing cells. Environ Inter 30:261–278 McLaughlin MJ, Parker DR, Clarke JM (1999) Metals and micronutrients-food safety issues. Field Crop Res 60:143–163 Merroun, ML (2007) Interactions between metals and bacteria: fundamental and applied research In: Méndez-Vilas A (ed) Communicating current research and educational topics and trends in applied microbiology, pp 108–119 Mohamed RM, Abo-Amer AE (2012) Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane. J Basic Microbiol 52:53–65 Nies DH (1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51:730–750 Nordberg G (2003) Cadmium and human health: a perspective based on recent studies in China. J Trace Elem Exp Med 16: 307–319 Peralta-Videa JR, Lopez ML, Narayan M, Saupe G, Gardea-Torresdey J (2009) The biochemistry of environmental heavy metal uptake by plants: Implications for the food chain. Int J Biochem Cell Biol 41: 1665–1677 Rico M, Lopez A, Santana-Casiano JM, Gonzalez AG, Gonzalez-Davila M (2013) Variability of the phenolic profile in the diatom Phaeodactylum tricornutum growing under copper and iron stress. Limnol Oceanogr 58:144–152
Roane TM, Josephson KL, Pepper IL (2001) Dual-bioaugmentation strategy to enhance remediation of co-contaminated soil. Appl Environ Microbiol 67:3208–3215 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor press, New York Schaller J, Weiske A, Mkandawire M, Dudel EG (2010) Invertebrates control metals and arsenic sequestration as ecosystem engineers. Chemosphere 79:169–173 Singh SK, Tripathi VR, Jain RK, Vikram S, Garg SK (2010) An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb Cell Fact 9:59 Sulaymon AH, Ebrahim SE, Mohammed-Ridha MJ (2013) Equilibrium, kinetic, and thermodynamic biosorption of Pb (II), Cr (III), and Cd (II) ions by dead anaerobic biomass from synthetic wastewater. Environ Sci Pollut Res 20:175–187 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729 Thomson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acid Res 22:4673–4680 Veglio F, Beolchini F (1997) Removal of metals by biosorption: A review. Hydrometall 44:301–316 Vieira DM, Augusto da Costa AC, Henriques CA, Cardoso VL, Pessoa de Franca F (2007) Biosorption of lead by the brown seaweed Sargassum filipendula—batch and continuous pilot studies. Elec J Biotech 10:369–375 Volesky B (1990) Biosorption of heavy metals. CRC Press, Boca Raton Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261– 5267 White C, Wilkinson SC, Gadd GM (1995) The role of microorganisms in biosorption of toxic metals and radionuclides. Intern Biodeterior Biodegrad 35:17–40 Yetis U, Dolek A, Dilek FB, Ozcengiz G (2000) The removal of Pb (II) by Phanerochaete chrysosporium. Water Res 34:4090–4100
RESEARCH ARTICLE
Biosequestration of lead using Bacillus strains isolated from seleniferous soils and sediments of Punjab Saurabh Gupta & Richa Goyal & Nagaraja Tejo Prakash
Received: 19 December 2013 / Accepted: 21 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The present study was conducted to isolate and explore bacterial strains with a potential to sequester lead (Pb) and tolerate other heavy metals from industrial effluents and sediments. Out of the six bacterial strains isolated from seleniferous sites of Punjab, three isolates (RS-1, RS-2, and RS-3) were screened out for further growth-associated lead sequestration and molecular characterization on the basis of their tolerance toward lead and other heavy metals. Biomass and cell-free supernatant were analyzed for lead contents using ICP-MS after growth-associated lead sequestration studies in tryptone soya broth (pH=7.2±0.2) under aerobic conditions at 37 °C temperature. Almost 82 % and 70 % divalent lead was sequestered in cell pellets of RS-1 and RS-3, respectively while only 45 % of lead was found in cell pellet of RS-2 in the first 24 h. However, significant biosequestration of lead was observed in RS-2 after 48 h of incubation with concomitant increase in biomass. Simultaneously, morphological, biochemical, and physiological characterization of selected strains was carried out. 16S rRNA gene sequence of these isolates revealed their phylogenetic relationship with class Bacillaceae, a low G + C firmicutes showing 98 % homology with Bacillus sp.
Responsible editor: Robert Duran S. Gupta (*) Department of Microbiology, Mata Gujri College, Fatehgarh Sahib 140406, Punjab, India e-mail: [email protected] R. Goyal Department of Microbiology, Dolphin PG College of Life Sciences, Chunni Kalan 140307, Punjab, India N. T. Prakash Department of Biotechnology & Environmental Sciences, Thapar University, Patiala 147004, Punjab, India
Keywords Metal sequestration . Biotransformation . Bioaccumulation . Lead
Introduction Heavy metals, metalloids, and radionuclides released from ores and industrial activities have become a major concern in terms of environmental pollution (Schaller et al. 2010). Accumulation of these metals/metalloids in different ecosystems leads to an enhanced risk to abiotic and biotic components of associated ecosystem (Luoma and Rainbow 2008). Leaching of these elements and their compounds into soil and ground water facilitate their easy uptake in plants followed by animals through food chains and imparts adverse effects on human health (Nordberg 2003). Even at low concentrations, heavy metals like mercury (Hg), cadmium (Cd), and lead (Pb) directly cause oxidative stress, lipid peroxidation, carcinogenesis, mutagenesis, and neurotoxicity in humans, animals, and plants (Ahmad et al. 2012; Gupta and Ali 2004; Howlett and Avery 1997; Jaroslawiecka and Piotrowska-Seget 2014). Lead is one of the most toxic pollutants in the environment absorbed by plants without any known biological function (Peralta-Videa et al. 2009). Elevated concentration of lead released into an ecosystem through smelting, mining, electroplating, and paint industry become a serious pervasive pollutant. Finally, this is transported to the biological system through ground water followed by its biomagnifications in living beings through the food chain (McLaughlin et al. 1999). Human exposure to higher concentrations of lead (Pb) may lead to renal tubular dysfunction along with proteinuria, glucosuria, and tubular necrosis (Alfven et al. 2002). Inhalation of lead results in metal fume fever with chemical pneumonitis, pulmonary edema, and death (Hayes 2007). The use of biological systems, especially plants and microorganisms, for removal of heavy metals from contaminated
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sites is a fast growing and promising technology with several advantages over traditional methods such as precipitation, filtration, ion exchange, oxidation reduction, electrochemical recovery, membrane separation, and other techniques (Dhankher et al. 2002). Microorganisms use various resistance mechanisms such as permeability barriers, intra- and extracellular sequestration, efflux pumps, enzymatic detoxification, and reduction to overcome heavy metal stress (Nies 1999). Numerous studies on biosorption of lead using different biological material have been reported in recent years. Different living systems such as marine algae, fungi, and yeasts possess significant metal-binding capacity for a wide range of heavy metals and metalloids (Al-Garni 2005; Deng et al. 2007; Halttunen 2007; Kogej et al. 2010; Vieira et al. 2007). In most of the studies dealing with the bio-removal of metals, either dead or resting biomass has been used (Deng et al. 2007; Gadd 1990). Bio sequestration is one of the bioremediation approach where application of a judicious consortium of growing metal-resistant cells ensure better removal through a continuous metabolic uptake of metals after physical adsorption. Bio sequestration may lead to the simultaneous removal of toxic metals, organic loads, and other inorganic impurities, as well as allow optimization through the development of resistant species (Malik 2004). However, there are limited reports on the Pb-sequestration using viable and aerobically growing bacteria. In the present study, three aerobic strains were isolated from selenium-contaminated sites with multiple metal tolerance along with lead to remediate lead contaminated sites. Besides India, selenium contaminated soils have been reported in China, South Africa, Columbia, Argentina, Venezuela, Spain, Bulgaria, Ireland, Algeria, Morocco, Australia, and New Zealand and in some drier regions of the former Soviet Union (Gupta et al. 2010). Present study reports one of the few observations on lead sequestration up to 50 mg/l with actively growing Bacillus sp.
Materials and methods Collection of samples Soil and sludge samples were collected from the Jainpur village of Hoshiarpur–Nawanshahr regions of Punjab in India (75° 55 E; 31° 56 N). Elevated selenium contents in low-lying areas of this region were reported where selenium oxyanions are transported by rain water through seasonal rivulets from the nearby hills of the Shivalik range and deposited in the low-lying areas. The toxic sites are located at the dead ends of the seasonal rivulets. Indigenous microbial communities undergo various self-recovery processes in these polluted sites. Microorganisms have evolved a variety of mechanisms to detoxify these heavy metals, and some even use them for respiration. Hence 1 kg of soil was collected two
times around the year in the months of March and October from these selected patches in sterile polythene bags from a depth of 0–15 cm. Samples were brought to laboratory and were used as such without any prior treatment for enrichment and isolation of lead tolerant bacteria. Five grams of sample was suspended in tryptone soya broth (TSB), pH=7.2±0.2, containing (per liter): 17-g peptone from casein, 3.0-g peptone from soy meal, 2.5-g glucose, 5.0-g NaCl, and 2.5-g K2HPO4 supplemented with lead as lead nitrate (lead as 5.0 mg/l a.i.). Flasks were incubated at 37 °C on an incubator shaker (120 rpm) until growth appeared. A small aliquot was then transferred to the fresh media with increased metal ion concentrations up to 10 mg/l for enrichment of lead tolerant bacteria. After sufficient sub-culturings, a loopful of culture was streaked on tryptone soya agar (TSA) plates to obtain axenic cultures. Plates were incubated under aerobic conditions at 37 °C for 24 h. Colonies with different morphology were picked in pure form and preserved on TSA plates at 4 °C for further studies. Multiple metal tolerance studies These bacterial isolates were further screened for their tolerance and resistance toward different metals. Axenic culture was inoculated in TSB (pH=7.2±0.2) supplemented with different concentrations (i.e., 5.0, 10.0, 25.0, and 50.0 mg/l as an active ingredient) of lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni), arsenic (As), tin (Sn), selenium (Se), zinc (Zn), chromium (Cr), and copper (Cu). Inoculated flasks containing media were incubated at 37 °C for 48 h in shaking conditions (120 rpm) and were observed for appearance of growth. Pb-impacted growth and sequestration studies Three bacterial isolates, namely RS-1, RS-2, and RS-3, were further explored to examine lead (Pb) stressed growth and sequestration studies. An aliquot of these isolates was inoculated in 100-ml TSB (pH =7.2 ± 0.2) supplemented with 50 mg/l lead as PbNO3 in the culture medium and incubated on an orbital shaker at 120 rpm and 37 °C. Simultaneously, a control lacking lead was also maintained for comparison. Initially, growth was monitored after regular interval of 6 h up to 24 h followed by measurement of growth after every 24 h up to 96 h. To check the metal sequestration in the biomass, an aliquot was withdrawn after regular interval of 24 till 96 h. These samples were centrifuged (8,000 rpm at 4 °C for 10 min) for separation of biomass and cell-free supernatant. Biomass and cell-free supernatants were acid digested as described elsewhere (Gupta et al. 2012). In short, biomass and supernatant were acid digested with pre-mixed concentrated nitric acid (HNO3) and perchloric acid (HClO4) in 3:1 ratio. Acid-digested samples
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were diluted with 0.2 % HNO3 and were subjected to inductively coupled plasma mass spectrometry (ICP-MS) analysis to determine distribution of lead in different fractions.
ICP-MS analysis ICP-MS analysis was carried out on PerkinElmer Sciex ELAN DRC-e (Axial Field Technology). The system was operated under inert conditions maintained with argon gas at the flow rate of 15 l through plasma, at 1.2 l and 0.9 l from auxiliary and nebulizer, respectively, throughout the measurement. Pressure was maintained between 60–70 psi. Plasma formation was carried out at 6,000 °C temperature which causes the sample to separate into individual atoms (atomization) by ionization with plasma and detection by mass spectrometer.
Characterization of the bacterial isolates Three selected strains RS-1, RS-2, and RS-3, were characterized in terms of their morphological and biochemical characteristics along with identification and phylogenetic relatedness with other organisms. Biochemical characterization of these strains was carried out at the Microbial Type Culture Collection (MTCC-IMTECH), Chandigarh, India. Molecular characterization was carried out by isolating genomic DNA using standard protocols (Sambrook et al. 1989), amplifying and sequencing the 16S ribosomal RNA (rRNA) gene. PCR amplification was carried out using 27 F and 1492R universal primers (Singh et al. 2010). PCR product was eluted, purified, and sequenced at DNA sequencing facility, Delhi University, Delhi. Sequences were analyzed by using CHECKCHIMERA program of the Ribosomal Database Project (RDP)-II (Maidak et al. 2001). 16S rRNA gene sequences of isolates were compared with those available in GenBank/ EMBL databases using the BLAST program (Altschul et al. 1997). The sequences of closely related strains and uncultured bacteria were retrieved from RDP-II and aligned using multiple alignments CLUSTAL-W program (Thomson et al. 1994; Cole et al. 2007). Phylogenetic dendrograms were constructed by neighbor-joining method in which associated taxa were clustered together by the bootstrap test using MEGA 6 package (Felsenstein 1985; Tamura et al. 2013).
Statistical analysis Metal tolerance, growth, and sequestration experiments were carried out in triplicate and results are presented as mean values along with standard error in the respective figures.
Results Dynamic genetic and biochemical capacity of microorganisms, especially bacteria, has introduced these organisms as an effective tool toward bioremediation of organic and inorganic pollutants such as heavy metals (Roane et al. 2001; Jaroslawiecka and Piotrowska-Seget 2014). In 2004, Malik reviewed the use of growing microorganisms for metal removal and development of technically and economically viable technologies for the treatment of metal-rich effluents. In this work, we have investigated potential of selected bacterial strains toward sequestration of lead for the restoration of naturally and anthropogenic heavy metals-contaminated sites which in turn will limit the transport of these heavy metals in the biological systems. As a part of this study, six lead tolerant bacterial strains were isolated from the selenium-contaminated soils and selected strains were explored for growth-associated lead sequestration along with tolerance of all the isolates for other heavy metals.
Multiple metal tolerance studies A wide variation in minimum inhibitory concentration (MIC) of different metals was observed for different isolates. Isolates RS-1, RS-2, RS-3, and RS-5 were invariably resistant to lead, nickel, and selenium up to 50 mg/l. While minimum inhibitory concentration (MIC) of other metals showed an intermittent range for these isolates (Table 1). RS-4 and RS-6 showed least tolerance for most of the metals with maximum resistance for selenium (50 mg/l) only. RS-5 has already been reported for mercury sequestration (Gupta et al. 2012) while RS-1, RS-2, and RS-3 were further explored in the present study for sequestration of lead.
Table 1 Heavy metal tolerance/MIC (mg/l) profile of different isolates Metals
Lead (Pb) Mercury (Hg) Cadmium (Cd) Nickel (Ni) Arsenic (As) Tin (Sn) Selenium (Se) Zinc (Zn) Chromium (Cr) Copper (Cu)
Isolates RS-1
RS-2
RS-3
RS-4
RS-5
RS-6
50 25 25 50 10 0 50 10 25 10
50 10 50 50 10 0 50 10 25 10
50 25 25 50 10 0 50 25 25 10
25 25 10 10 0 0 50 10 10 5
50 50 25 50 25 0 50 50 50 50
10 0 0 10 0 0 50 10 10 5
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Growth-associated Pb sequestration studies Effect of lead was studied on selected bacterial strains (RS-1, RS-2, and RS-3) in terms of their growth in leadsupplemented culture medium. Growth of these isolates was monitored as increase in optical density over a period of 96 h with concomitant increase in biomass (Fig. 1). The isolates showed typical growth pattern in both control (without lead) and lead (Pb)−containing media. An insignificant lag phase has been observed in RS-1 and RS-3 (Fig. 1a, c) with little effect of lead on starter culture. Besides this, limited effect of lead was observed in subsequent growth phases of RS-1 and RS-3 as indicated by comparable growth in both control and lead supplemented media during stationary phase (Fig. 1a, c). In RS-2, comparatively longer generation time was observed in lead-supplemented media as compared to all other isolates as indicated by optical density measurement (Fig. 1b). Similar slow growth rate was also observed in control of RS-2 with respect to other isolates (Fig. 1). In metal sequestration studies, profound sequestration of metal was observed in biomass of RS-1 followed by RS-3 during the first 24 h as indicated by amount of lead quantified in the cell pellet as compared to the cell-free supernatant (Fig. 2a, c). Approximately, 82 % of metal accumulation was observed in RS-1 followed by 70 % metal sequestration in RS-3. RS-2 showed comparatively less metal sequestration during the first 24 h of incubation (Fig. 2b). Only 45 % of metal was accumulated in the biomass that corroborates with the relatively slow growth of RS-2. Even after prolonged incubation of RS-1 and RS-3 up to 96 h, there was no further significant increase in sequestration of lead observed. On the contrary, substantial increase in biosequestration of lead was observed in RS-2 up to 48 h. After 96 h of incubation, a slight increase of lead was observed in cell-free supernatant of all the isolates (Fig. 2). Characterization and identification of isolates These bacterial strains (RS-1, RS-2, and RS-3) were characterized in terms of their morphological, physiological, and biochemical characteristics. All the isolates were invariably rod shaped and stained Gram-positive, were motile, spore
forming, and did not produce any pigment. Physiologically, RS-3 tolerated a wide range of pH (5.0–11.0), temperature (15–45 °C) and a high salt concentration up to 10 %w/v followed by an intermediate range with RS-1 and a narrow range by RS-2 (Table 2). Out of these three isolates, RS-1 and RS-3 were found catalase and oxidase positive with no nitrate reduction while RS-2 exceptionally reduced nitrate and was oxidase negative. Analysis of the 16S rRNA gene sequence by multiple sequence alignment (BLAST) indicated 99 % homology of all the three strains to Bacillus sp. with 98 % alignment coverage over ~1.5 kb. The sequence was assigned the GenBank accession number HM179550, HM179551, and HM179552. Classification of these isolates to genera Bacillus was confirmed using the RDP-II Classifier (Wang et al. 2007) and evolutionary history was inferred using the maximum composite likelihood method (Fig. 3).
Discussion Remediation of heavy metals-contaminated sites through common physico-chemical techniques is expensive and not eco-friendly. Hence, the use of biotechnological approaches such as use of different microorganisms has received an immense attention as alternative tool (Iyer et al. 2005). A lot of work has been published about the genetic and biochemical mechanisms that microorganisms use to withstand metal toxicity with descriptions of how microorganisms physiologically adapt to metal stress (Chen et al. 2013; Sulaymon et al. 2013; Jaroslawiecka and Piotrowska-Seget 2014). No single strategy has been adopted by microorganisms for survival under heavy metals-stressed conditions. A number of diverse and complex mechanisms of resistance and adaptation such as reduced uptake of toxic metal, efflux, sequestration, metabolic bypass, and chemical modifications have been reported (Gonzalez et al. 2010; Lemire et al. 2013; Rico et al. 2013). Biosorption is a process in which dead and inactive biomass retain relatively high quantities of heavy metals including lead by passive sorption and/or complexation (Veglio and Beolchini 1997; Yetis et al. 2000; Deng et al. 2007). However, the low-binding capacity of biomass for certain recalcitrant metals and failure to effectively remove metals
Fig. 1 Comparative growth kinetics of bacterial strains with and without Pb. 1a RS-1; 1b RS-2; 1c RS-3
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Fig. 2 Comparative bioaccumulation of Pb in different bacterial strains. 2a RS-1; 2b RS-2; 2c RS-3
Table 2 Morphological, physiological, and biochemical characteristics of the RS-1, RS-2, and RS-3
Characters Morphological Cell shape Gram stain Motility Capsule Colony morphology Pigmentation Physiological Growth at 4 °C Growth at 42 °C Growth at 45 °C Growth with 1 % NaCl Growth with 5 % NaCl Growth with 7 % NaCl Growth with 10 % NaCl Growth at pH 2 Growth at pH 5 Growth at pH 8 Growth at pH 11 Biochemical characteristics Indole utilization Methyl red Voges Proskeur Citrate utilization Nitrate reduction H2S production Urease Starch hydrolysis Casein hydrolysis Lipid hydrolysis Gelatin liquefaction
NA nutrient agar
Oxidase Catalase Carbohydrate fermentation test Sucrose Glucose Lactose
RS-1
RS-2
RS-3
Rod shape Gram + ve + − Circular, flat, undulate margins −
Rod shape Gram + ve + − Large, flat, opaque, irregular, undulate margins −
Rod shape Gram variable + + Smooth, undulate margin Yellow/ white
− − − + +
+ − − + +
− + + + +
− − − − + +
− − − + + −
+ + − + + +
− + + − −
− − + + +
− + + + −
− − + + + +
− − + + + +
− − − + + +
+ +
− +
+ +
+ − +
− + −
+ + ±
Environ Sci Pollut Res Fig. 3 Evolutionary relationships of RS-1, RS-2, and RS-3 with other taxa using the neighborjoining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method
from real industrial effluents limit this approach. Hence, the use of actively growing metal-resistant cells ensures better removal through a combination of bioprecipitation, biosorption, and continuous metabolic uptake of metals after physical adsorption (Malik 2004; Volesky 1990). Previously, a number of bacteria have been reported for uptake and transformation of heavy metals and metalloids such as mercury, cadmium, and selenium (Gupta and Jashan 2014; Kumar et al. 2012; Dhanjal and Cameotra 2010). Similarly, in the present study, growth-associated uptake of lead in the biomass has been reported. This observation was in corroboration with growth of the cultures. Growth-associated metal tolerance has been recently reported in Pseudomonas aeruginosa with no data on bioaccumulation capacity
(Mohamed and Abo-Amer 2012). Certain other bacteria such as Alcaligenes faecalis, P. aeruginosa, and B. iodinium were reported with a potential to remove Pb in growth-associated metal detoxification studies (Jaysankar et al. 2008). Growth and metabolism-dependent metal removal involve metal precipitation as sulfides, complexation by siderophores and other metabolites, sequestration by metal-binding proteins and peptides, transport and intracellular compartmentation (White et al. 1995). Bacillus sp. has been observed to form surface layer (S-layer) protein that can be used as a template for sequestration of heavy metals from the industrial effluent (Merroun 2007). Extracellular polymeric substances (EPS), secreted by bacteria, results in the formation of biofilms, which facilitates in situ metal sequestration under pilot scale
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(Edwards and Kjellerup 2013). Exopolysaccharides secreted by Halomonas sp. have net negative charge due to high uronic acids content and play a vital role in sequestration of heavy metals (Gutierrez et al. 2012). In this study, sequestration of lead was carried out in complex medium with high content of dissolved organic carbon (DOC) which might have played an important role in the sequestration of lead. Initial slow growth of RS-2 with concomitant less sequestration of lead may be attributed to inherited property of this bacterial strain toward DOC present in medium. While speciation of intracellular lead was not performed in the present study, lead uptake and transformation was quite evident. Formation of lead sulfide was speculated from change in biomass color from cream to black (data not shown). Over 96 h, the profile of total lead in various fractions viz., cell-free supernatant and biomass indicated that the organism diverts further lead uptake by its expulsion in the supernatant (may be immobilized form) although the expelled fraction is always much less than the accumulated fraction. Since all the isolates grow well in significantly high concentrations of lead and other heavy metals with the potential to sequester lead inside the biomass, these findings indicate efficient detoxification and transformation mechanisms harnessed by these organisms for high concentration of lead metal/cation.
Conclusion This study thus presents salient findings on growth-associated lead sequestration by Bacillus strains isolated from seleniferous sites of Punjab. Exploitation of these strains toward the accumulation of heavy metals within the biomass may provide a useful approach for remediating lead contamination present in various industrial effluents. Interestingly, this is the first report on viable and actively growing Bacillus strains capable of Pb ion sequestration.
References Ahmad E, Zaidi A, Khan MS, Oves M (2012) Heavy metal toxicity to symbiotic nitrogen-fixing microorganism and host legumes. In: Zaidi A, Wani PA, Khan MS (eds) Toxicity of Heavy Metals to Legumes and Bioremediation. Springer Verlag Wein, New York, pp 29–44 Alfven T, Jarup L, Elinder CG (2002) Cadmium and lead in blood in relation to low bone mineral density and tubular proteinuria. Environ Health Perspect 110:699–702 Al-Garni SM (2005) Biosorption of lead by Gram-negative capsulated and non-capsulated bacteria. Water SA 31:3 Altschul SF, Thomas LM, Alejandro AS, Zhang J, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acid Res 25:3389–3402
Chen J, Qin J, Zhu Y, Lorenzo V, Rosen BP (2013) Engineering the soil bacterium Pseudomonas putida for arsenic methylation. Appl Environ Microbiol 79:4493–4495 Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohidden AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tieje JM (2007) The ribosomal database project (RDP-II): introducing my RDP space and quality controlled public data. Nucl Acid Res 35(Database issue):D169–D172 Deng L, Su Y, Su H, Wang X, Zhu X (2007) Sorption and desorption of lead (II) from wastewater by green algae Cladophora fascicularis. J Haz Mat 143:220–225 Dhanjal S, Cameotra SS (2010) Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil. Microb Cell Fact 9:52 Dhankher OP, Li Y, Rosen BP, Shi J, Salt D, Senecoff JF, Sashti NA, Meagher RB (2002) Engineering tolerance and hyper accumulation of arsenic in plants by combining arsenate reductase and gammaglutamylcysteine synthetase expression. Nat Biotechnol 20:1140– 1145 Edwards S, Kjellerup BV (2013) Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals. Appl Microbiol Biotechnol 97:9909–9921 Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791 Gadd GM (1990) Heavy metal accumulation by bacteria and other microorganisms. Experientia 46:834–840 Gonzalez AG, Shirokova LS, Pokrovsky OS, Emnova EE, Martinez RE, Santana-Casiano JM, Gonzalez-Davila M, Pokrovski GS (2010) Adsorption of copper on Pseudomonas aureofaciens : Protective role of surface exopolysaccharides. J Coll Inter Sci 350:305–314 Gupta VK, Ali I (2004) Removal of lead and chromium from waste-water using bagasse fly ash—a sugar industry waste. J Colloid Interface Sci 271:321–328 Gupta S, Nirwan J (2014) Evaluation of mercury biotransformation by heavy metal-tolerant Alcaligenes strain isolated from industrial sludge. Int J Environ Sci Technol. doi:10.1007/s13762-013-0484-9 Gupta S, Prakash R, Tejoprakash N et al (2010) Selenium mobilization of Pseudomonas aeruginosa (SNT-SG1) isolated from seleniferous soils from India. Geomicrobiol J 27:35–42 Gupta S, Goyal R, Nirwan J, Cameotra SS, Tejoprakash N (2012) Bio sequestration, transformation and volatilization of mercury by Lysinibacillus fusiformis isolated from industrial effluent. J Microbiol Biotechnol 22:684–689 Gutierrez T, Biller DV, Shimmield T, Green DH (2012) Metal binding properties of the EPS produced by Halomonas sp. TG39 and its potential in enhancing trace element bioavailability to eukaryotic phytoplankton. Biometals 25:1185–1194 Halttunen T (2007) Removal of lead, cadmium and arsenic from water by lactic acid bacteria. Dissertation, Department of Biochemistry and Food Chemistry, University of Turku, Turku Hayes AW (2007) Principles and methods of toxicology. Taylor & Francis press, Philadelphia Howlett NG, Avery SV (1997) Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation. Appl Environ Microbiol 63:2971–2976 Iyer A, Mody K, Jha B (2005) Biosorption of heavy metals by a marine bacterium. Mar Poll Bull 50:340–343 Jaroslawiecka A, Piotrowska-Seget Z (2014) Lead resistance in micro-organisms. Microbiology 160:12–25 Jaysankar D, Ramaiah N, Vardanyan L (2008) Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar Biotechnol 10:471–477 Kogej A, Likozar B, Pavko A (2010) Lead Biosorption by selfimmobilized Rhizopus nigricans pellets in a laboratory scale packed
Environ Sci Pollut Res bed column: mathematical model and experiment. Food Technol Biotechnol 48:344–351 Kumar A, Gupta S, Cameotra S (2012) Screening and characterization of potential cadmium biosorbent Alcaligenes strain from Industrial effluent. J Basic Microbiol 52:160–166 Lemire JA, Harrison JJ, Turner RJ (2013) Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Microbiol 11:371–384 Luoma SN, Rainbow PS (2008) Metal contamination in aquatic environments: science and lateral management. Cambridge University Press, Cambridge Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM (2001) The RDP-II (ribosomal database project). Nucl Acid Res 29:173–174 Malik A (2004) Metal bioremediation through growing cells. Environ Inter 30:261–278 McLaughlin MJ, Parker DR, Clarke JM (1999) Metals and micronutrients-food safety issues. Field Crop Res 60:143–163 Merroun, ML (2007) Interactions between metals and bacteria: fundamental and applied research In: Méndez-Vilas A (ed) Communicating current research and educational topics and trends in applied microbiology, pp 108–119 Mohamed RM, Abo-Amer AE (2012) Isolation and characterization of heavy-metal resistant microbes from roadside soil and phylloplane. J Basic Microbiol 52:53–65 Nies DH (1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51:730–750 Nordberg G (2003) Cadmium and human health: a perspective based on recent studies in China. J Trace Elem Exp Med 16: 307–319 Peralta-Videa JR, Lopez ML, Narayan M, Saupe G, Gardea-Torresdey J (2009) The biochemistry of environmental heavy metal uptake by plants: Implications for the food chain. Int J Biochem Cell Biol 41: 1665–1677 Rico M, Lopez A, Santana-Casiano JM, Gonzalez AG, Gonzalez-Davila M (2013) Variability of the phenolic profile in the diatom Phaeodactylum tricornutum growing under copper and iron stress. Limnol Oceanogr 58:144–152
Roane TM, Josephson KL, Pepper IL (2001) Dual-bioaugmentation strategy to enhance remediation of co-contaminated soil. Appl Environ Microbiol 67:3208–3215 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor press, New York Schaller J, Weiske A, Mkandawire M, Dudel EG (2010) Invertebrates control metals and arsenic sequestration as ecosystem engineers. Chemosphere 79:169–173 Singh SK, Tripathi VR, Jain RK, Vikram S, Garg SK (2010) An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb Cell Fact 9:59 Sulaymon AH, Ebrahim SE, Mohammed-Ridha MJ (2013) Equilibrium, kinetic, and thermodynamic biosorption of Pb (II), Cr (III), and Cd (II) ions by dead anaerobic biomass from synthetic wastewater. Environ Sci Pollut Res 20:175–187 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729 Thomson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acid Res 22:4673–4680 Veglio F, Beolchini F (1997) Removal of metals by biosorption: A review. Hydrometall 44:301–316 Vieira DM, Augusto da Costa AC, Henriques CA, Cardoso VL, Pessoa de Franca F (2007) Biosorption of lead by the brown seaweed Sargassum filipendula—batch and continuous pilot studies. Elec J Biotech 10:369–375 Volesky B (1990) Biosorption of heavy metals. CRC Press, Boca Raton Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261– 5267 White C, Wilkinson SC, Gadd GM (1995) The role of microorganisms in biosorption of toxic metals and radionuclides. Intern Biodeterior Biodegrad 35:17–40 Yetis U, Dolek A, Dilek FB, Ozcengiz G (2000) The removal of Pb (II) by Phanerochaete chrysosporium. Water Res 34:4090–4100
