Elucidate the Influence of Heavy Metal on Bacterial Growth Isolated from a Mining Location and A Waste Dump: Using their Inducible Mechanism
Sarita Sharma, Rathod Zalak R and Saraf Meenu S*
Department of Microbiology and Biotechnology, University School of Sciences, India
Submission: October 1, 2021; Published: October 25, 2021
*Corresponding author: Saraf Meenu S, Department of Microbiology and Biotechnology, University of School of Sciences, Gujarat University Ahmedabad-380009, Gujarat (India)
How to cite this article: Sarita Sharma, Rathod Zalak R, Saraf Meenu S. Elucidate the Influence of Heavy Metal on Bacterial Growth Isolated from a Mining Location and A Waste Dump: Using their Inducible Mechanism. Curr Trends Biomedical Eng & Biosci. 2021; 20(2): 556034. DOI:10.19080/CTBEB.2021.20.556034
Abstract
The main aim of this study was to analyze the mobilization of storage reserves during seed germination of Citrus limon by host plant-endophytic bacteria interaction and to determine the contribution of endophytic bacteria in plant establishment. The seeds were inoculated with three different endophytic bacteria from Bacillus spp. which were isolated from the leaf of Citrus limon plant, and they were germinated in the dark. Fresh weight changes and early seedling growth were assessed, in all germinated and non-germinated seeds. To understand the mechanism promoting seed germination, the activities of extracellular enzymes of bacterial isolates were also analyzed by the plate assay method. The results showed that treatment with endophytic bacteria accelerated during the germination process. In summary, our current study showed that endophytic bacteria improved seed germination and post-germination seedling growth of Citrus limon plant inoculation with endophytic bacteria could promote storage reserve mobilization during or following germination; three bacterial isolates might have different action mechanisms on seed germination. The improved germination percentage and decreased germination period (GPD) using external agents can be exploited in conservation of this overexploited medicinally important plant.
Keywords: Citrus limon; Endophytic bacteria; Seed germination; Reserve mobilization
Introduction
The accumulation of hazardous metals from ore tailings dumping and leachate overflow in and around mining areas is currently creating concerns about environmental stress. Mining activities, particularly open-pit mining, often produce the most solid wastes in the form of waste rock. Acidic mine drainage (AMD) discharged with high amounts of heavy metals can contaminate downstream water, agricultural soils, food crops, and biota, posing a health concern to populations living near mines [1-3]. Mining-related heavy metal contamination is a serious global environmental hazard, particularly in developing countries [4]. According to a Times of India storey about a municipal solid waste dumpsite, the Pirana Landfill site takes approximately 4000 tonnes of rubbish per day, with the majority of it being deposited in the landfill untreated. Furthermore, hazardous chemical waste and by-products generated by a number of industries/factories in the vicinity of the landfill site were dumped in a haphazard manner [5]. Because of their high toxicity, cadmium and lead are considered among the main contaminants [6,9]. Mine tailings, effluents from the textile, leather, tannery, electroplating, and galvanising industries, as well as cadmium batteries, all discharge cadmium into the environment [10]. Furthermore, cadmium is a reasonably common element that is not present in its pure form in nature. Steel plating, stabilizers for polyvinyl chloride colours in plastics and glassware, electrode material in nickel–cadmium batteries, and as a component of various alloys and ceramics are some of its main applications [11]. At low concentrations, heavy metals such as Ni, Co, and Pb, however, cause oxidative stress, lipid peroxidation, carcinogenesis, mutagenesis, and neurotoxicity in people, animals, and plants [12]. The discharge of heavy metal-containing effluents puts pressure on the ecosystem, posing health risks to plants, animals, aquatic life, and humans. Toxic metals are transferred to groundwater and bioaccumulated after surface contamination [13,14]. When certain amounts of heavy metals are introduced into the environment, the bulk of the micro flora dies, leaving only a few cells with metal resistance mechanisms. Heavy metals can cause significant changes in microbial populations and their activity when they are introduced into the environment in various forms [15]. Several research have looked into the heavy metal sensitivity or resistance of bacteria isolated from various habitats, as well as their processes for adapting to the hazardous metal during exposure [16,17]. Some microbes have been reported to have evolved heavy metal detoxification systems, and some even exploit them for respiration, making them resistant to them [18]. Permeability barriers, intracellular and extracellular sequestration, efflux pumps, enzymatic detoxification, and reduction are some of the inducible mechanisms used by microbes to cope with heavy metal stress [19]. The use of heavy metal-tolerant rhizobacteria to boost metal bioavailability in heavy metal-polluted soil is a promising strategy. The goal of this work was to extract and characterize rhizobacteria that tolerate Cadmium, Lead, and Nickel from heavy metal-polluted soil. Isolated heavy metal tolerant rhizobacteria could be useful as bioremediation agents and in conjunction with phytoremediation of Cd2+, Pb2+, and Ni2+ contaminates in soil.
Material and Methods
Bacterial culture
A total of 51 rhizosphere bacteria were recovered from the Zawar mines in Udaipur, Rajastha [20], while 40 rhizosphere bacteria were obtained from the Pirana trash site in Ahmedabad, Gujarat. The bacterial colonies SMHMZ2, SMHMZ4, and SMHMZ46 from the Zawar mines, as well as SMHMP4, SMHMP23, and SMHMP38 from the Pirana landfill site, showed better resistance to heavy metals (Ni2+, Pb2+, and Cd2+).
Chemicals and media used
The media employed in this investigation came from Hi Media Laboratories in Mumbai, India, and includes nutrient agar compositions such Peptone, Beef extract, Yeast extract, and Agar powder. NaCl, (NH4)2SO4, K2HPO4, KH2PO4, CaCl2, FeSO4, MgSO4, and glucose were furnished by Fine Chemicals (P) Ltd. in New Delhi, India. Heavy metal standards such as NiCl2.7H2O, Pb (NO3)2, and CdCl2 were given by SRL Pvt. Ltd., Mumbai, India.
Heavy metal stock solution preparation
We tested the tolerance of 91 bacterial isolates obtained from rhizosphere soil from mines and landfill sites to three heavy metals Ni2+, Pb2+, and Cd2+at various concentrations. Ni2+ (5000µg/ml), Pb2+ (5000µg/ml), and Cd2+ (5000µg/ml) heavy metal stock solutions were prepared using NiCl2.7H2O, Pb (NO3)2, and CdCl2, respectively. All of the above-mentioned heavy metal solutions were sterilised by autoclaving for 15 minutes at 15 psi pressure and 121⁰C [20]. The growth rates of all six isolates were assessed using growth cubes in the presence and absence of respective heavy metals. To begin each culture’s growth curve, two types of inoculums were used.
Development of the inoculum
Inoculum with metal (WM) was created with 3 mL Nutrient medium and 1% culture, as well as the above-mentioned metal concentration, at a temperature of 37⁰C and 150rpm on a spinning shaker, with pH 7. As a result, Ni2+, Pb2+, and Cd2+ inoculums have been developed for each culture. To increase each culture’s adaptability to heavy metals (in this case, Ni2+, Pb2+, and Cd2+) in Nutrient medium, we repeated the technique and circumstances for inoculum development with metals four times for all cultures. All six cultures’ inoculum WM in Ni2+, Pb2+, and Cd2+ entered log phase within 2-6 hours in the subsequent four transfers. In subsequent transfers, the time it takes for each culture to reach the log phase has decreased. 3 mL Nutrient medium and 1% culture were used to make inoculums without metal (W/OM). In this situation, metal-free inoculums have been established for all cultures. In the absence of heavy metals, all cultures reached their exponential phase within 2-12 hours.
Growth Studies
To assess the ideal growth conditions in terms of pH and temperature, all bacteria isolates were grown in nutrient medium with a pH of 7, at a temperature of 370C and 150rpm on a spinning shaker. Following the development of both types of inoculums, the growth curves of each culture were started using 1% inoculum WM & W/OM in 200ml nutrient medium containing the desired metal concentrations Ni2+ (1500 ppm), Pb2+ (1000 ppm), and Cd2+ (500 ppm), against which we had to check the tolerance of each culture. At various time intervals after the start of the development curve for each culture, OD values at 600 nm spectrophotometrically (Shimadzu, Model No.1722) were acquired. As a result, for each of the six cultures, we have two growth curves for each metal, one starting with inoculum generated WM and the other starting with inoculum W/OM.
Result and Discussion
In the presence of all three metals Ni2+, Pb2+, and Cd2+ the lag phase of bacterial cultures SMHMZ2, SMHMZ4, SMHMZ46, SMHMP4, SMHMP23, and SMHMP38 is reduced when the growth curve is started with inoculum developed in the presence of metal, compared to growth curves started with inoculum developed without metal, where the lag phase is increased. As a result, the findings show that inducible proteins play a role in the tolerance mechanisms displayed by bacterial isolates SMHMZ2, SMHMZ4, SMHMZ46, SMHMP4, SMHMP23, and SMHMP38. Inoculums formed in the lack of metal induced the extended lag phase in the development curve. As a result, the metal acts as an inducer, causing bacteria to make inducible proteins that help them evolve and tolerate their environment. The lag period is minimized in the cases of bacterial isolates SMHMZ2, SMHMZ4, SMHMZ46, SMHMP4, SMHMP23, and SMHMP38 because inoculums created with metal after four subsequent transfers became adapted due to the establishment of an inducible mechanism towards heavy metal tolerance. After inoculation into new Nutrient media, their development commences rather quickly (with a shorter lag period) due to a previously described inducible mechanism.
Conclusion
In polluted soil samples from a mining location and a waste dump site, we discovered 91 rhizosphere bacterial species, according to our research. Six bacterial strains with potential in heavy metal resistance and bioremediation were identified after testing on greater concentrations of cadmium, lead, and nickel. The growth kinetics showed that bacterial cultures SMHMZ2, SMHMZ4, SMHMZ46, SMHMP4, SMHMP23, and SMHMP38 were more tolerant. As a result, the metal acts as an inducer in the culture, resulting in the synthesis of inducible proteins and a protein inducible mechanism in the SMHMZ2, SMHMZ4, SMHMZ46, SMHMP4, SMHMP23, & SMHMP38, lowering the lag phase even in the presence of significant heavy metal concentration.
Acknowledgement
For encouragement and assistance with required facilities, I am grateful to my Guide Professor (Dr.) Meenu Saraf, Head of Department of Microbiology and Biotechnology and Director of University School of Sciences, Gujarat University, Gujarat, India, and Faculty member of Department of Microbiology and Biotechnology, Gujarat University, Gujarat, India. I’d like to express my gratitude to the Gujarat State Education Department’s SHODH (Scheme of Development High Quality Research) for presenting me with a scholarship.
References
- Tian D, Zhu F, Yan W, Fang X, Xiang W, et al. (2009) Heavy metal accumulation by panicled goldenrain tree (Koelreuteria paniculata) and common elaeocarpus (Elaeocarpus decipens) in abandoned mine soils in southern China. J Environ Sci (China) 21: 340-345.
- Duruibe JO, Ogwuegbu MOC, Egwurugwu JN, (2007) Heavy metal pollution and human biotoxic effects. Int J Phys Sci 2: 112-118.
- Plumlee GS, Morman SA (2011) Mine wastes and human health. Elements 7: 399-404.
- Zhuang P, Lu H, Li Z, Zou B, McBride MB (2014) Multiple exposure and effects assessment of heavy metals in the population near mining area in South China. PLoS One 9: e94484.
- Joshi H, Jadeja R, Zala B, Belani N, Chauhan A (2019) Study of heavy metal contamination in soil surrounding pirana landfill site by atomic absorption spectrometer. 5(4): 6.
- Salinas E, Elorzade Orellano M, Rezza I, Martinez L, Marchesvky E, et al. (2000) Removal of cadmium and lead from dilute aqueous solutions by Rhodotorula rubra. Bioresource Technol 72: 107-112.
- Blaudez D, Botton B, Chalot M, (2000) Cadmium uptake and sub cellular compartmentation in the ectomy corr hizal fungus Paxillusin volutus. Microbiology 146: 1109-1117.
- Carrillo Gonzalez R, Gonzalez Chavez Mdel C, (2012) Tolerance to and accumulation of cadmium by the mycelium of the fungi Scleroderma citrinum and Pisolithus tinctorius. Biol Trace Elem Res 146: 388-395.
- Jaeckel P, Krauss GJ, Krauss G (2004) Cadmium and zinc response of the fungi Heliscus lugdunensis and Verticillium cf. alboatrum isolated from highly polluted water. Sci Total Environ 346: 274-279.
- Woldeamanuale TB (2017) Isolation, Screening and Identification of Cadmium Tolerant Fungi and Their Removal Potential. J Forensic Sci & Criminal Inves 5(1):
- Govil PK, Sorlie JE, Murthy NN, Sujatha D, Reddy GLN, et al. (2008) Soil contamination of heavy metals in the Katedan Industrial Development Area, Hyderabad, India. Environ Monit Assess 140:313-323.
- Joseph B, George J, Jeevitha MV (2012) Impact of heavy metals and Hsp response. Int J Biosci 2(9): 51-64.
- Mohiuddin K, Ogawa Y, Zakir HM, Otomo K, Shikazono HW, et al. (2009) Characteristics on soil heavy metals pollution around mine waste piles. Int Conf Environ Sci Inf Appl Technol.
- Santhaveerna Goud, Mahendra BG (2010) Influence of soil characteristics on bioremediation of hydrocarbons contaminated contaminated soil. J Environ Res Develop 4(3): 734-741.
- Sheik CS, Mitchell TW, Rizvi FZ, Rehman Y, Faisal M, et al. (2012) Exposure of soil microbial communities to chromium and arsenic alters their diversity and structure. PLoS ONE 7(6): e40059.
- Rajbanshi A (2008) Study on heavy metal resistant bacteria in Guheswori sewage treatment plant. Our Nature 6(1): 52-57.
- Thippeswamy B., Sivakumar CK, Krishnappa M (2012) Acumulation potency of heavy metals by Saccharomyces Species indigenous to paper mill effluent. J Environ Res Develop 6(3): 439-445.
- Ezaka E, Anyanwa CU (2011) Chromium (VI) Tolerance of bacterial strains isolated from sewage oxidation ditch. Int J Environ Sci 1(2): 1725-1734.
- Joshi BH, Modi KG (2013) Screening and characterization of heavy metal resistant bacteria for its prospects in bioremediation of contaminated soil. Journal of Environmental Research and Development 7(4A): 1531-1538.
- Sharma S, Shah RK., Rathod ZR, Jain R, Lucie KM, et al. (2020) Isolation of Heavy Metal Tolerant Rhizobacteria from Zawar Mines Area, Udaipur, Rajasthan, India. Bioscience Biotechnology Research Communication Special Issue 13(1): 233-238.