Evaluation of Lead resistance by newly identified Enterobacter cloacae strain AHZ 01 isolated from contaminated soil

Greener Journal of Biological Sciences

Vol. 10(2), pp. 42-47, 2020

ISSN: 2276-7762

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Evaluation of Lead resistance by newly identified Enterobacter cloacae strain AHZ 01 isolated from contaminated soil

Aisami A^1,*; Mahdi Z.A¹; Chindo H.A¹; James, I.J; Garba, L²

¹Department of Biochemistry, Faculty of Sciences, Gombe State University, P.M.B. 027, Gombe, Nigeria.

²Department of Microbiology, Faculty of Science, Gombe State University, P.M.B. 027, Gombe, Nigeria.

ARTICLE INFO	ABSTRACT
*Article No.:* 0622520080 *Type: Research*	Heavy metal pollution is currently one of the major environmental challenges worldwide. Lead among the leading heavy metal pollutant. Removal of pollutants through physical and chemical method can also generate secondary pollution but bio-removal of metal pollutants from industrial soil and wastewater by microbes resistant to heavy metal is a fundamental aspect of environmental biotechnology. In this current study, a bacterial was isolated from contaminated soil by using nutrient broth incorporated with Pb(NO3)2. The isolate was identified by gram staining and molecular approach and the effect of lead concentration and temperatures were determined. The isolate is a gram-positive and identified as Enterobacter cloacae strain AHZ 01. The isolates were subjected to different Pb(NO3)2, the results revealed that 85 % of the 50 ppm was removed within 96 hours. The result also showed that 75 %, 67 %and 50 % of 100, 150, and 200 ppm respectively were removed within 96 hours. While for 250 ppm only 20 % was removed after 96 hours, but 250 ppm showed an inhibitory effect with a slower growth rate and less than 20 % was removed. A 35 ℃ was the best temperature for the removal of the Lead ions while 40 and 45 ℃. The r RNA sequence of the isolate was deposited at NCBI Genbank and accession number of MN880497 and the bacterial can remove 85 % of 50 ppm Pb(NO3)2. The isolate is a good candidate for further bioremediation studies.
*Accepted:* 25/06/2020 *Published:* 28/07/2020
Corresponding Author Dr Abubakar Aisami* *E-mail:* Abubakar.aisami05@ gmail.com *Phone:* +234806775052
*Keywords: Heavy metals; Lead; rRNA; Enterobacter cloacae; pollution*.

1.0 INTRODUCTION

Heavy metals are normal constituents of the environment, but their increased industrial discharges processes result in their accumulation and eventually polluting the environment. Bioremediation is known to be one of the healthier, cleaner, cost-effective and environmentally friendly methods for the decontamination of polluted areas with a wide variety of contaminants. The term bioremediation was used to explain the mechanism of extracting hazardous waste from the atmosphere using a biological agent. Bioremediation is the most effective method for handling the toxic environment and reclaiming degraded land. Bioremediation cycle utilizes different agents including bacteria, fungi, algae and higher plants as key instruments for the removal of heavy metals in the ecosystem (Aisami et al. 2016).

Bacterial to resistance heavy metals occur naturally in the environment from bacterial contact with the metals. However, rigorous human action and exploitation of natural deposits have led to an increased number of metal-resistant micro-organisms (Bruins et al., 2000). Heavy metals pollution of the is on the increase, and negatively affects both plants and animals (Gaur et al., 2014). The bioaccumulation of heavy metals such as Mn, Zn, Mg, Cu, Cd, Ni, and Pb in the environment is a major warning to human life (Hooda, 2007). Lead is considered as the most severe environmental pollutant among several other metals and metalloids (Sparks, 2005). Heavy metals pollution can cause diseases and disorders, even in relatively low concentrations (Osibote et al. 2016). Industrially activities, for example, the production of batteries, pigments and metal smelting, as well as the manufacturing of products such as lead arsenate insecticides or lead water pipes, are the significant ways through which Pb gets into the environment. Mobilisation of lead from minerals and natural processes such as volcanic emission and soil erosion are minor contributors to the environments (Gadd, 2010; Yokel and Delistraty, 2003). The average lead concentration in the soils is from 10 to 100 mg/kg while industrial areas can be up to 10000 mg/kg (Akmal and Jianming, 2009; Schwab et al., 2005).

Furthermore, In the industrial wastewaters, the level of Pb (II) scopes from 200–250 mg/l, however, according to conventional quality standards, it should not surpass 0.05–0.10 mg/l (Sag et al., 1995). As a consequence of human actions, like the burning of fossil fuel, mining, and lead-containing compounds manufacturing, can be found in all parts of our environment (Suparna et al., 2011).

Heavy metals are cytotoxic at low concentrations and will cause cancer in humans (Dixit et al., 2015). Heavy metals are containing industrial effluent that causes health hazards to plants, animals, aquatic life, and humans are increasing pressures on flora and fauna (Robin et al., 2012). Heavy metals are incredibly hazardous and present within the environment beyond their limits (Zeeshanur and Ved Pal, 2016).

The physic-chemical means of getting rid of heavy metals from the environment such as precipitation, oxidation-reduction, ion exchange, filtration, and reverse osmosis, among others (Ahluwalia and Goyal, 2005) rescind the ecosystem and produces secondary pollution. Bioremediation can be used to remove the heavy metal pollutants that are injurious to living (Mandal et al., 2012). The use of microorganisms to remediate these heavy metals is both cheaper and environmentally friendly than conventional methods such as precipitation. This treatment is more effective than any other technique (Alavijeh et al., 2014). Many Pb (II) resistant microbes to have been isolated from soils contaminated with metals, water wastes and industrial wastes (Jarosławiecka and Piotrowska-Seget, 2014). This study aimed to isolate, identify, and characterised the lead(Pb) resistant-resistance bacteria.

2.0 MATERIAL AND METHODS

Preparation of Heavy metal stock and working solutions

Stock solutions of lead (Pb) 1000 ppm was prepared by dissolving 1.6 g of lead nitrate Pb(NO₃)₂ in 1 litre distilled water. The working solutions 50, 100, 150, 200 and 250 ppm were prepared from the stock solution by diluting with the accurate volume of distilled water.

2.1 Sample collection and isolation of lead resistant bacteria

A soil sample was collected from the contaminated area at Bolari, Gombe State, Nigeria in sterilised polythene bags and it was preserved at 4^oC until it was used for bacteria isolation (One gram of the soil sample was added to 9ml of sterilised water in test tubes; 1 ml of each sample suspension was spread on the Nutrient agar supplemented with seven (7ppm) lead nitrate (Pb (NO₃)₂) and incubated at room temperature for 24hrs and growth was observed Makki et al., 2019). A colony was sub-cultured on a nutrient agar plate and incubated for 24hrs at 37 ◦C (Al-Haik et al., 2016). A pure culture was obtained and was kept on nutrient agar inside a Bijou bottle at room temperature for analysis. The isolate was named as AHZ 01.

2.2 Gram staining

The cells from overnight nutrient broth culture were prepared. A drop from the prepared cells was dropped on a clean glass slide and spread with a loop over to cover the surface of the glass slide; then, it was allowed to dry out. The slide was exposed with methanol and allowed to evaporate at room temperature. Heat-fixed smear slides were placed on staining tray and were flooded the smear was then gently submerged with crystal violet and allowed for 1 minute. The slide was then tilted slightly and gently washed with distilled water, then gently flooded with gram’s iodine and permitted to stand for 1 minute. The smear slide was rinsed with distilled water using a wash bottle. Decolourization was done using 95% ethyl alcohol by applying the dropwise for 10 seconds and rinsed instantly with water. Safranin used to counter-stain and stand for 1 minute and rinsed with distilled. It was viewed by the use of a light microscope under oil immersion.

2.3 Molecular

Genomic DNA was extracted from Pellets from a 24 h grown nutrient broth bacterial cells by the utilisation of bacterial using genomic DNA extraction protocol (Thermo Scientific GeneJet Genomic DNA Extraction) instruction.

PCR amplification of 16S rRNA gene

The amplification of the 16S rRNA was done using polymerase chain reaction (PCR) using universal primers, 27f 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R: 5'-TAC GGT TAC CTT GTT ACG ACT T-3' (Aisami et al. 2020) as forward and reverse primers respectively with genomic DNA as the template. 25 µL PCR reaction mixture which composed of 1µL forward primer, one µL reverse primers, 1 µL DNA template, 9.5 µL master mix, 12.5 µL deionised water was prepared. The polymerase chain reaction (PCR) was conducted under the following conditions: 1 cycle of initial denaturation at 96ºC for 4 min; 30 cycles (of denaturation for 1 min, 52.3ºC annealing for 1 min, 72ºC extension for 1 min and one cycle of final extension at 72ºC for 7 min. Presence of the amplified 16S rRNA gene was confirmed using 1.0% agarose gel electrophoresis (Fermentas, USA) and viewed with UV transilluminator (UPV, USA).

2.4 Analysis of the 16S rRNA gene and construction of the phylogenetic tree.

The sequences were matched using the BLAST program (http://www.ncbi.nlm nih.gov/BLAST) for the identification of the bacterial isolate.

2.5 Growth Study of Metal Resistant Isolates

The ability of the bacterial strain for the tolerance of heavy metal was established by growing the bacterial cells in a 250 ml nutrient broth (NB) by adding different concentrations of lead and lead, incubated for 24, 48, and 72 hrs. At 37°C on a rotary shaker (150 rpm). The Growth was examined as a function of biomass by measuring absorbance at 600 nm using a spectrophotometer. Bacteria cultured in NB without lead supplementation served as control. Estimation of Pb concentration was done using Atomic absorption spectroscopy (AAS) by centrifuging the grown cells at 10,000 rpm for 10 min as a method described by Baghel (2016).

Adsorption of metals with bacterial cells was calculated as a ratio of metal removal %.

R (%) = (C0-C1)/ C0 * 100.

Where R is removal Ratio (%)

C0 is the concentration of heavy metals in the original solution (µg/ml) and

C1 is the concentration of heavy metal in the treated solution (µg/ml) (Qin et al., 2006).

2.6 Effect of temperature on Bacterial resistance to lead.

The lead resistant isolate was inoculated into 250ml of nutrient broth incorporated with different lead concentrations as and incubated at different temperatures (20, 25, 30 35 40 and 45, ◦C) for 24 hours. Bacterial growth was recorded by measuring the absorbance using a spectrophotometer for three consecutive days. The concentration of heavy metal was also recorded after four days using Atomic Absorbance Spectrophotometer.

Adsorption of metals with bacterial cells was calculated as a ratio of metal removal %.

R (%) = (C0-C1)/ C0 * 100.

Where R is removal Ratio (%)

C0 is the concentration of heavy metals in the original solution (µg/ml) and

C1 is the concentration of heavy metal in the treated solution (µg/ml) (Qin et al., 2006).

3.0 Results and Discussion

3.1 Gram stain

The gram staining result of isolate AHZ 01 revealed that cells were red, demonstrating a characteristic of Gram-negative. It is rod-shaped. The microscopic analysis exemplifies that the colony is creamy white, as shown in Figure 1.

4. 0 CONCLUSION

Enterobacter cloacae strain AHZ 01 was isolated from contaminated soil and identified, it tolerates up to 200 ppm lead nitrate and growth parameters such as temperature and concentration used. The temperature of 35 ^oC is the best temperature for the growth of the bacteria. The bacteria is a good microorganism for remediation in the tropical area.

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