EJBR2020v10i4art326 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2020; 10(4): 326-335 DOI: http://dx.doi.org/10.5281/zenodo.4023161 Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B Amina Musa Jibrin1, Oluwafemi Adebayo Oyewole1*, Japhet Gauis Yakubu1, Aisha Hussaini1, Evans Chidi Egwim2 1 Department of Microbiology, Federal University of Technology Minna, Niger State, Nigeria 2 Department of Biochemistry, Federal University of Technology Minna, Niger State, Nigeria *Correspondence author: E-mail: oa.oyewole@futminna.edu.ng Received: 08 June 2020; Revised submission: 10 August 2020; Accepted: 27 August 2020 http://www.journals.tmkarpinski.com/index.php/ejbr Copyright: © The Author(s) 2020. Licensee Joanna Bródka, Poland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: Biosorption is the ability of biological materials to accumulate heavy metals from wastewater through mediated or physico-chemical pathways of uptake. Urease producing bacteria have been hypothesized to have inherent bioremediation abilities. The aim of this research was to determine the potential of Lysinibacillus fusiformis 5B to biosorp Pb, Cr, Cd and Ni. The stock solution of Pb, Cr, Cd and Ni was prepared by dissolving 0.0157 g of Pb(C2H3O2)2, 0.057 g of K2Cr2O7, 0.018 g of CdSO4 and 0.026 g of NiSO4 in 100 mL of dH2O respectively. Lysinibacillus fusiformis 5B was screened for the potential to utilise 5 ppm of the heavy metals using agar dilution method. Broth of L. fusiformis 5B was inoculated to 10, 15, 20 and 50 ppm of the heavy metals. The rate of biosorption was determined by atomic absorption spectroscopy (AAS) after 0, 7, 14, 21, 28 and 35 days. The biosorption % was determined by Beer Lambart’s equation. Lysinibacillus fusiformis 5B was able to tolerate 5 ppm concentration of all the heavy metals by showing visible growth on surfaces of nutrient agar Petri plates. Generally, there was an increase in biosorption rate as the days progress. After 35 days of incubation, the highest biosorption rate of 99.96%, 99.97%, and 99.94% were recorded for Pb, Cr, and Cd respectively at 10 ppm and 99.33% of Ni at 15 ppm. The results of this study showed that L. fusiformis 5B possess the capacity to biosorp Pb, Cr, Cd and Ni and can be developed as biosorption agent for these heavy metals. Keywords: Lysinibacillus fusiformis 5B; Biosorption; Cadmium; Chromium; Lead; Nickel. 1. INTRODUCTION Increase in human civilization and active involvement in industrialization have caused improvements in the standard of living of humans across the globe. However, the industrialization has led to the release of harmful chemical substances, which have now become an environmental problem affecting both plants, animals and man [1]. Some of these harmful substances enter the environment as solid, liquid and gaseous wastes [2] from anthropogenic activities, which have greatly contributed to the abundant presence of heavy metals in the environment [2-5]. Aside anthropogenic activities, heavy metals can also be released naturally into the environment via volcanic eruption and the weathering of metal-bearing rock particles [2, 6-7]. These heavy metals have been Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 327 European Journal of Biological Research 2020; 10(4): 326-335 reported to deter physiological functions of biological systems [1]. Metals whose densities are greater than 5 g/cm3 are categorized as heavy metals. These heavy metals have atomic number greater than 20 and are poisonous and toxic at low concentrations [3]. Some heavy metals (i.e. Cu, K, Cr, Fe, Zn, Ni, Na, Mg and Mn) serve as essential micronutrient required by biological systems in small concentration to stabilize molecules through interactions that are electrostatic [3, 8-9]. They are also required for various redox processes and the regulation of osmotic pressure as well as a component required for a functional enzyme system [8]. Whereas other heavy metals (i.e. Al, Pb, Cd, Au, Hg, Ag) are non-essential and have no biological function. They are toxic and a potential threat to all biological system [8-11]. Human exposure to these heavy metals above the World Health Organization safe level published by Olawale [12] and Calderón et al. [2] could lead to adverse health effects such as; nausea, skin allergy, fluorosis of skin, indigestion, diarrhea as well as cancer. Other health impacts include neuronal damage, chronic asthma, kidney and liver damage, cardiovascular disorders, teratogenicity, mutations, congenital disorders, impairment of sensory nerves, chronic anemia, memory loss, depression, autoimmune diseases, mood swings, anxiety, drowsiness, hair loss, fatigue, blindness, brain damage and insomnia [4, 8, 12-14]. These among many more health disorders associated with heavy metal exposure have caused scientists and research bodies all over the world to search for novel technologies to alleviate this environmental threat. Conventional methods involving physical or chemical processes have been and are still in use in alleviating heavy metal polluted environment [12]. Some of these conventional methods include; membrane filtration, chemical precipitation, floatation, electrodialysis, reverse osmosis, solvent extraction, photocatalysis, ion exchange, electrochemical treatment, microfiltration, ultrafiltration and nanofiltration [11, 13-15], seem effective but cause other environmental problems such as destruction of soil structure, generation of secondary pollutants, which are resistant to other cleaning treatments, expensive to install as well as time consuming [16-18]. These limitations have made researchers to exploit biological means. The use of biological materials in remediating an environment polluted by heavy metals is known as bioremediation [11, 19]. Bioremediation is inexpensive and above all, environmentally friendly. It could either be ex-situ or in-situ through biomineralization, bioaccumulation, bioleaching, biotransformation or biosorption of the heavy metals [1, 5, 13, 20-21]. Biosorption have emerged as the most promising technology among other bioremediation techniques, which involves a passive uptake mechanism and is most times reversible and is not dependent on metabolism of the biological material but rather the surfaces of the materials, which acts in the sorbent of heavy metals [1, 2, 12, 20]. Biosorption could either be carried out by sorbent materials from biological means, which could be from plants and most times from microbial source [1, 11, 23]. The use of microorganisms in biosorption of heavy metals have received great attention due to fact that they can be sourced cheaply, with high effective adsorption capacity, are reusable, and could utilize both living and dead cells since it only involves the cell wall of the microorganisms [3, 11]. Many factors influence the capacity of microorganisms to adsorb metals from the environment, which include; microbial status (age of cell), properties of the metal ions (valence, radius among others), biosorption conditions (i.e. temperature, pH, contact time, concentration of microbial biomass and metals, presence of other ions, micronutrition and metal ions availability) and culture conditions (composition of growth media, nutrition supply and carbon source) [11, 19]. Among microorganisms (fungi, yeast, algae and bacteria) [24] involved in biosorption, though fungi are effective but bacteria have emerged as the most promising with fast growing rate and a wide range of binding sites. Bacteria have over time evolved and created specific genes Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 328 European Journal of Biological Research 2020; 10(4): 326-335 that help them to survive in environment contaminated with heavy metals. Most used bacteria genera in alleviating environmental pollution include; Bacillus and Pseudomonas owing to their high binding affinity for heavy metals [4]. The functional groups present on bacteria surfaces such as sulfonate, carboxyl, hydroxyl, phosphonate, amide and sulfonate are mainly utilized in active metal uptake from the environment [1]. The presence of these functional groups on bacterial surfaces makes it possible for binding of metals through ionic binding of metal cations involving electrostatic forces. Structure of bacteria cell has made it possible to be distinguished as either Gram positive or Gram negative. Cell wall of Gram positive bacteria is thicker than that of the Gram negative bacteria because of the thick peptidoglycan and presence of teichuronic and teichoic acids [25]. This is however opposite of Gram negative bacteria, which have a thin peptidoglycan without teichuronic and teichoic acids. This characteristic has made Gram positive bacteria to be most efficient in adsorption of heavy metals [1]. Lysinibacillus fusiformis is Gram positive, rod-shaped, spore forming, non-motile bacteria belonging to the Bacillaceae family. It is referred to as Lysinibacillus due to the presence of Asp-Lys type of peptidoglycan in the cell wall. Varying bacterial species have been engineered such that their enzymes play an essential role in biomineralization of heavy metals [26]. This is true about ureolytic bacteria such as L. fusiformis capable of producing urease, which hydrolyses urea to give ammonia and carbon dioxide [27]. Ureolytic bacteria are capable of forming bonds with heavy metals to form minerals as an important path during biogeochemical cycles of elements [28]. In these biogeochemical cycles, calcium carbonate produced during hydrolysis of urea causes the precipitation of soluble heavy metals in what is known as microbial induced minerals precipitation (MIMP). During MIMP, bacterial cells can hold heavy metals and radionuclides within their cells by adsorption and/or coprecipitation of the heavy metals in lattice of calcite [29]. Lysinibacillus fusiformis have been previously reported by varying literatures on their prowess potential in the sequestration, intracellular transformation, precipitation and volatilization of chromate [30], boron [31], mercuric chloride [32], cadmium and copper [23], magnesium and calcium ions [33]. As such, the aim of this research was to biosorp lead, chromium, cadmium and nickel by urease producing L. fusiformis 5B. 2. MATERIALS AND METHODS 2.1. Sample collection The bacterial strain L. fusiformis 5B used in this study was collected from the laboratory of Department of Microbiology, Federal University of Technology Minna, Nigeria. The isolate was reported to have the ability to produce urease. The isolate was subcultured into newly prepared nutrient agar (NA) so as to get a fresh culture for the study. The purity of the strain was confirmed by Gram staining and viewing under the microscope at ×100 objectives lenses. 2.2. Preparation of heavy metal solutions The stock solution of nickel sulfate and cadmium sulfate was prepared by dissolving 0.026 g and 0.018 g respectively into 100 mL of distilled water. Whereas 0.057 g of potassium dichromate and 0.0157 g of lead acetate was measured and dissolved into 100 mL of distilled water to get their respective stock solutions. Agitation was carried out on the stock solutions for 15 minutes and allowed to stand for 24 h to ensure complete dissolution of metal salts. Atomic Absorption Spectrophotometry (AAS) was used to measure the initial concentration of metal solutions (Ni, Cd, Pb and Cr). The pH of heavy metal solutions was also adjusted using sodium hydroxide (NaOH) and hydrochloric acid (HCl) to a pH of 7 [6]. Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 329 European Journal of Biological Research 2020; 10(4): 326-335 2.3. Heavy metals tolerance of Lysinibacillus fusiformis 5B Heavy metal tolerance by L. fusiformis 5B was ascertained using agar dilution method. Concentration of 5 ppm of the heavy metals (lead, chromium, nickel and cadmium) was prepared and incorporated into nutrient agar before sterilizing using the autoclave at 121°C for 15 minutes. The modified media were all allowed to cool down to 40°C before dispensing into their respective well labelled Petri dishes and allowed to solidify. From a 24 h culture broth of L. fusiformis 5B, a sterile swab stick was used to aseptically inoculate into the different heavy metal Petri plates by swabbing gently on the surfaces of the media. The culture plates were then incubated at 37°C for 24 h in an inverted position. Development of bacterial colonies indicates the ability of the isolate to tolerate the heavy metal while absence of visible colonies indicates that the test organisms were unable to tolerate the heavy metals [18]. 2.4. Biosorption of heavy metals The heavy metal nutrient broth culture medium was prepared into different concentrations (10, 15, 20 and 50 ppm) using the prepared stock solutions. The culture broth containing the varying concentration of heavy metals was then sterilized at 121°C for 15 minutes, after which the culture broth was allowed to cool before inoculating 5 mL of 24 h old culture, where cells of L. fusiformis 5B have attained 1.5 ×106 cfu/mL with the exception of the blank, which was used as control. The heavy metal culture broths were incubated aerobically in an incubator with shaker at 37°C for 35 days. 2.5. Wet digestion for the determination of Total Cd, Pb, Ni and Cr using Atomic Absorption Spectroscopy The 0.2-0.5 grams of sample was weighed into a 100 mL volumetric flask, 30 mL of wet digestion acid (650 mL of nitric acid in 1 L beaker, 80 mL of perchloric acid and 20 mL of sulfuric acid) and stirred to mix. Sample was placed on a fume cupboard and digested until sample reduce to 20 mL. The heating was continued until white fumes of nitric acid disappeared and sample reduced to 10 mL. The sample was transferred quantitatively to a 50 mL volumetric flask and made to mark with dH2O. It was then shaken vigorously and filtered through a Whatman 0.45 µ m filter paper. A 1 mL of the clear digest was pipetted into another 50 mL volumetric flask and made to mark with dH2O. Samples were read using AAS (AA WIN 500 PG instrument) at 7 days interval starting with zero reading (day 1) using wavelengths 359.4 nm, 326.1 nm, 283.3 nm and 231.1 nm for chromium, cadmium, lead and nickel respectively. The percentage of biosorption was determined by measuring the amount of heavy metal removed from the medium through estimation of the residual metal concentration using AAS. Beer Lambert’s law (Equation 1) was used to achieve the percentage biosorption [6]. % Biosorption = [(Initial metal concentration – final metal concentration) / Initial metal concentration] x 100 Equation 1 2.6. Data analysis Statistical package for social science (SPSS 24) utilizing one-way analysis of variance (ANOVA) was used to analyze the data generated from this study. 3. RESULTS 3.1. Heavy metal tolerance of Lysinibacillus fusiformis 5B Lysinibacillus fusiformis 5B was able to tolerate concentration of 5 ppm of all heavy metals (Ni, Cd, Cr Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 330 European Journal of Biological Research 2020; 10(4): 326-335 and Pb) by showing visible growth of abundant colonies on surfaces of heavy metals nutrient agar Petri plates (Figure 1). Figure 1. Growth of Lysinibacillus fusiformis 5B in 5 ppm of heavy metals. 3.2. Biosorption of lead by Lysinibacillus fusiformis 5B The result obtained from the biosorption of lead by L. fusiformis 5B at different concentrations and at different time intervals is shown in Table 1. A general increase in the absorption of lead was observed across all concentration observed (5, 15, 20 and 50 ppm). Within the first seven days of incubation, the highest rate (70.44 %) of biosorption was recorded in 20 ppm and the least (40.06 %) was recorded for 50 ppm. After the 35 days of incubation, the highest biosorption (99.96%) of lead was recorded in 10 ppm and the least (86.61 %) was recorded for 50 ppm. Table 1. Biosorption percentage of lead by L. fusiformis 5B. Lead concentration (%) Days 10 15 20 50 7 58.01 ± 0.01d 52.36 ± 0.36d 70.44 ± 0.44 e 40.06 ± 0.06 e 14 76.54 ± 0.54c 69.87 ± 0.87 c 74.44 ± 0.44 d 50.11 ± 0.11 d 21 97.40 ± 0.40b 94.68 ± 0.68 b 78.11 ± 0.11 c 57.29 ± 0.29 c 28 99.80 ± 0.80a 99.41 ± 0.41 a 88.14 ± 0.14 b 70.14 ± 1.14 b 35 99.96 ± 0.96 a 99.89 ± 0.89 a 99.24 ± 0.24 a 86.61 ± 0.61 a Values are x̄±SEM of duplicate values. x̄ with dissimilar letter(s)s are not significantly different from each other according to Duncan Multiple Range Test (DMRT). Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 331 European Journal of Biological Research 2020; 10(4): 326-335 3.3. Biosorption of Chromium by Lysinibacillus fusiformis 5B The biosorption of chromium by L. fusiformis 5B at different concentration and at different time intervals is presented in Table 2. After seven (7) days of incubation, a high rate of biosorption was recorded across all concentration with the highest (75.23%) recorded at 20 ppm and the lowest (55.69%) being 50 ppm. This high amount of biosorption of chromium was also recorded after fourteen (14) days. This however declined after 28 days of incubation. At the end of 35 days, biosorption of chromium was highest (99.97%) at 10 ppm and lowest (91.26%) at 50 ppm. Table 2. Result showing biosorption percentage of chromium by L. fusiformis 5B. Chromium concentration (%) Days 10 15 20 50 7 67.33 ± 0.33c 64.58 ± 0.58 d 75.23 ± 0.23 c 55.69 ± 0.69 e 14 95.95 ± 0.95b 83.33 ± 0.33 c 89.01 ± 0.01 b 66.62 ± 0.62 d 21 99.87 ± 0.87a 97.29 ± 0.29 b 98.96 ± 0.96 a 70.47 ± 0.47 c 28 99.92 ± 0.92a 99.34 ± 0.34 a 99.86 ± 0.86 a 86.72 ± 0.72 b 35 99.97 ± 0.97a 99.86 ± 0.86 a 99.93 ± 0.93 a 91.26 ± 0.26 a Values are x̄±SEM of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to Duncan Multiple Range Test (DMRT). 3.4. Biosorption of nickel by Lysinibacillus fusiformis 5B Biosorption of nickel by L. fusiformis 5B at different interval and concentration is represented in Table 3. Biosorption of nickel was recorded across all concentration and was highest (46.81%) at 20 ppm and lowest (20.99%) at 15 ppm. After 35 days of incubation, the biosorption was recorded highest (98.13%) at concentration of 10 ppm and the lowest (84.2 %) at 50 ppm. Table 3. Result showing biosorption percentage of nickel by L. fusiformis 5B. Nickel concentration (%) Days 10 15 20 50 7 37.91 ± 0.91d 20.99 ± 0.99d 46.81 ± 0.81d 24.60 ± 0.60e 14 66.27 ± 0.27c 54.72 ± 0.72c 64.14 ± 0.14c 38.40 ± 0.40d 21 90.47 ± 0.27b 78.27 ± 0.27b 82.49 ± 0.49b 44.90 ± 0.90c 28 92.89 ± 0.96b 96.96 ± 0.96a 80.49 ± 0.49b 64.90 ± 0.90b 35 98.13 ± 0.33a 99.33 ± 0.33a 91.70± 0.07a 84.24 ± 0.22a Values are x̄±SEM of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to Duncan Multiple Range Test (DMRT). 3.5. Biosorption of cadmium by Lysinibacillus fusiformis 5B The biosorption of cadmium by L. fusiformis 5B at different concentration and interval is recorded in Table 4. The highest biosorption rate (99.94%) recorded after 35 days of incubation was observed at 10 ppm whereas the lowest (97.23%) was recorded at 50 ppm. However, after day 7 of incubation, biosorption rate was highest (60.03%) and the lowest (44.31%) was recorded at concentration of 10 ppm. Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 332 European Journal of Biological Research 2020; 10(4): 326-335 Table 4. Result showing biosorption percentage of cadmium by L. fusiformis 5B. Cadmium concentration (%) Days 10 15 20 50 7 44.31 ± 0.31c 53.59 ± 0.59e 54.43 ± 0.43e 60.03 ± 0.03e 14 83.54 ± 0.54b 79.73 ± 0.73d 68.49 ± 0.43d 63.56 ± 0.56d 21 99.53 ± 0.53a 86.94 ± 0.94c 76.29 ± 0.29c 65.29 ± 0.29c 28 99.74 ± 0.74a 94.42 ± 0.42b 91.89 ± 0.89b 82.16 ± 0.16b 35 99.94 ± 0.94a 98.79 ± 0.79a 99.77 ± 0.77a 97.23 ± 0.23a Values are x̄±SEM of duplicate values. x̄ with dissimilar letter(s) are not significantly different from each other according to Duncan Multiple Range Test (DMRT). 4. DISCUSSION Bacterial cells have been reported in the past to possess inherent ability to survive in an environment polluted by varying contaminants such as petroleum and heavy metals [33]. Their survival have been attributed to their ability to respond adequately to stress from the environment through production of extracellular substances such as enzymes, fatty acids as well as polysaccharides making researchers to search for such microorganisms in an environment filled with heavy metal contaminants [34], among which bacteria genera such as Bacillus, Micrococcus, Streptomyces, Pseudomonas and Lysinibacillus have shown great potentials [24]. In this study, heavy metal tolerance was exhibited by L. fusiformis 5B against 5 ppm concentration of tested heavy metal salts. This was ascertained by the presence of abundant growth on the surfaces of cultured nutrient agar. This is however possible, owing to the components of the cell wall of Lysinibacillus species, which contains thick peptidoglycan, teichuronic and teichoic acid bonded by Asp-Lys [1]. Lysinibacillus species also have a mechanism that helps them actively pump out toxic substances from their cells in what is known as efflux pumps. Extracellular and intracellular sequestration of metal ions as well as reduction in membrane permeability are also strategies used by Gram positive bacteria to resist entry of toxic metal substances into their cells [31, 35]. He et al. [29] reported Lysinibacillus fusiformis ZC1 to be highly resistant to chromium. L. fusiformis ZC1 showed highest resistance reported so far for chromium as it recorded minimum inhibitory concentration of 60 mM. Likewise studies by Mathivanan et al. [23], which reported high heavy metal tolerance of L. fusiformis KMNTT-10 to lead (II) up to a concentration of 500 ppm. Biosorption of heavy metal carried out by L. fusiformis 5B in this study was observed across all concentrations (i.e. 10, 15, 20 and 50 ppm). After 7 days of incubation, the result obtained showed high rate (> 40%) of biosorption of heavy metals (Cd, Cr and Pb) across the concentration considered with the exception of nickel (Ni), which showed as low as 20.99% (15 ppm) and the highest at day 7 being 46.81% (20 ppm). This could be as a result of varying degree of toxicity of different heavy metal. In the biosorption of heavy metals by bacteria cells, the amount of time in which the bacterial cells are in contact with the heavy metal play a key role in biosorption. This was observed in this study, as the longer time the cells of L. fusiformis 5B were in contact with the heavy metal solution, the more the cells adsorb the heavy metal onto their cells. L. fusiformis 5B recorded low biosorption of Ni after the seventh day and a high rate of biosorption (>50%) across all concentration (10, 15 and 20 ppm) with the exception of 50 ppm. This shows that nickel may be more toxic to L. fusiformis 5B or the affinity of the functional groups present on the cell wall of L. fusiformis 5B was less compared to other heavy metals [24]. Jibrin et al. Heavy metals biosorption by urease producing Lysinibacillus fusiformis 5B 333 European Journal of Biological Research 2020; 10(4): 326-335 A high biosorption rate (> 55%) of chromium was recorded at day 7. This shows that the functional groups present on cell surface of L. fusiformis 5B have high affinity for ions of chromium present in the solution, which is in line with the observation made by He et al. [29] using L. fusiformis ZC1. It is important to note that biosorption of heavy metal reduces with increase in the concentration of the heavy metals (Cr, Ni and Pb). This is evident in this study, as at day 21, a high rate of biosorption (>90%) was recorded across all the heavy metals at concentration of 10 ppm. However, in the case of biosorption of cadmium in this study is not in correspondence with the general notion that the higher the heavy metal concentration the lower the biosorption as the result obtained for cadmium at day 7 of incubation of L. fusiformis 5B showed lowest biosorption (44.31%) at 10 ppm whereas the highest biosorption (60.03%) was recorded at 50 ppm. This could be related to the affinity the functional groups present on the surface of L. fusiformis 5B have on the metal ions since they all have binding sites, which could either be inhibited or enhanced at varying concentration of the heavy metal. This study observed little percent increase in the biosorption of metals by L. fusiformis 5B towards the latter stages of incubation. 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