Plant growth-promoting properties of metal-tolerant bacteria isolated from battery wastes dumpsites

Abstract

Environmental pollution by heavy metals arising from several industrial processes is a major cause for concern globally as these metals disrupt the soil ecosystem and rendering metal-polluted soils unfit for use in agriculture. As such, it is essential to devise means to reclaim these soils to sustain the soil biodiversity and enhance plant growth. This study focused on the determination of plant growth-promoting features of metal-tolerant bacteria (MTB) isolated from metal-polluted soils. Composite soil samples were obtained from three different sites receiving battery wastes. Using atomic absorption spectrophotometry, concentrations of heavy metals (cadmium, nickel, lead, zinc) in the soil samples were determined. Nutrient agar augmented with 50 μg/mL salts of the metals were used to cultivate MTB. Recovered MTB were screened for metal tolerance (100-500 μg/mL). Isolates demonstrating elevated multiple metal tolerance were identified using biochemical tests and identity confirmed using 16S rRNA sequencing, followed by screening for presence of metal-resistance genes (zntA, nreB, cadA, pbrA) using PCR and selected plant growth-promoting (PGP) traits. Statistical analysis of data generated was conducted using descriptive statistics. Of all contaminants, lead was most abundant (2596.8 ± 4.48 mg/kg). A total of 132 MTB were cultivated in pure culture, with 20 (15.15%) demonstrating multi-metal tolerance belonging to the genera Serratia (1), Citrobacter (1), Bacillus (1), Staphylococcus (2), Providencia (2), Mammaliicoccus (2), Pseudomonas (4) and Alcaligenes (7). Genes (pbrA/cadA/zntA) were detected (1/5/12). Nitrogen fixation/potassium/phosphate solubilisation/IAA/siderophores production were demonstrated by 15/10/5/13/17 isolates. Isolates obtained from the different sites exhibited significant multi-metal tolerance and demonstrated significant plant growth features.

Introduction

Industrial activities such as mining, smelting, and metal forging are major contributors to heavy metal contamination in soils. The accumulation of these pollutants can have detrimental impacts on soil ecology and overall soil health, which in turn negatively influence soil fertility and agricultural productivity [1]. Commonly detected heavy metals in contaminated soils include chromium, zinc, cadmium, mercury, nickel, and copper [2].

The acceleration of soil contamination by toxic heavy metals has been largely attributed to the widespread use of metals such as chromium, zinc, copper, cadmium, and lead in industrial operations including tanning, mining, refining, and manufacturing [3]. In addition, the application of heavy metals through fungicides, chemical fertilizers, wastewater irrigation, and sewage sludge has contributed significantly to the contamination of both water bodies and agricultural soils [4]. Heavy metals are non-biodegradable and tend to bioaccumulate in living organisms, thus pose a major and long-term ecological threat [5]. The release of heavy metals into the environment can significantly alter microbial communities and disrupt their functional activities. These metals typically exert toxic effects on microorganisms by inhibiting key biological processes, through mechanisms such as binding to essential functional groups, displacing vital metal ions, or altering the active conformations of biomolecules [6].

Since they serve as crucial co-factors for metalloproteins and enzymes, low quantities of specific transition metals, including cobalt, copper, nickel, and zinc, are necessary for numerous bacterial cellular functions [7-8]. Higher levels of these metals, however, are frequently harmful. Lead, cadmium, mercury, silver, and chromium are among the other heavy metals that are harmful even at low doses and have no known positive effects on bacterial cells [9]. Therefore, heavy metal remediation is required to shield the environment from the harmful impacts of these metals [10].

Meanwhile, a number of conventional techniques has been utilised in the clean-up of metal-polluted soils, but they leave the soils even more damaged than they were originally, and also not economically viable [11]. However, research efforts have been geared towards the application of microbes from metal-polluted soils with metal-detoxification potential as well as enhancement of plant growth, hence, tagged plant growth-promoting bacteria (PGPB) [12]. Indeed, PGPB enhance the growth of plants by various features including nitrogen fixation, solubilisation of phosphate and potassium, indole-3-acetic acid (IAA), siderophores, hydrogen cyanide and ammonia production [13]. Plant growth promoting bacteria (PGPB) also enhance plant competitiveness and responses to external stressors such as drought and metal contamination [14].

Thus, the aim of the current study is to determine the plant growth-promoting properties of metal-tolerant bacteria isolated from battery waste sites, in order to assess their potential for on-site metal removal and plant growth enhancement.

Materials and Methods

Collection of sample and quantification of zinc, cadmium, lead and nickel concentrations

Composite top soil samples were collected into well-labelled, sterile re-sealable bags from three different locations (A (7° 29' 01" N, 4° 04' 23" E), B (7° 29' 33" N, 4° 04' 11" E) and C (7° 29' 00" N, 4° 05' 00" E) within Ibadan metropolis (Figure 1). Samples were transported to the laboratory in icepacks for metal analysis and bacterial isolation. Extraneous materials from the soils were removed and oven-dried at 90 ℃ for 24 hours, after which concentrations of lead, cadmium, zinc and nickel were determined by initial digestion of the soils using hydrochloric acid and nitric acid (1:1), followed by subjection to atomic absorption spectrophotometry (Buck Scientific, Model 210 VGP) [15-16].

Figure 1.

Map of Lagelu local government area, Ibadan, Oyo State showing the various locations of sampling (in green).

Isolation of metal-tolerant bacteria

Composite top soil samples from the study site were subjected to serial dilution. Nutrient agar prepared using manufacturer’s instructions and appropriately sterilised, was supplemented with 50 μg/mL of filter-sterilised salts of zinc, lead, cadmium and nickel, separately and dispensed in sterile Petri dishes. Upon cooling and solidification, 1 mL from appropriate diluent was dispensed into the plates and incubated at 37 ± 2 ℃ for 24-48 hours [17]. Resulting colonies were considered as metal-tolerant bacteria (MTB). Pure bacterial culture was obtained by intermittent streaking of the isolates on sterile metal-supplemented nutrient agar plates [16].

Screening of MTB on increasing metal concentrations

The MTB were subjected to increasing concentrations of selected metal salts up to 500 μg/mL at intervals of 100 μg/mL Isolates that grew at a starting concentration was re-inoculated on higher concentration until growth stopped, this concentration was regarded as the minimum inhibitory concentration (MIC) [18].

Biochemical and 16S rRNA identification of MTB

Tentative identification of MTB exhibiting elevated multi-metal tolerance was done using appropriate morphological and biochemical characterisation [19-20]. Confirmatory identification was done through 16S rRNA sequencing of extracted bacterial DNA, with generated sequences subjected to BLAST against sequences available in the GenBank (National Centre for Biotechnology Information) [21].

Metal resistance-encoding genes detection in MTB

DNA of bacterial isolates were extracted via standard methods and subjected to PCR to amplify zinc-, cadmium-, lead- and nikckel-resistance genes. Primers targeting cadA, pbrA, zntA and nreB were utilised in the PCR process (Table 1) [22-25]. Conditions for PCR followed standard protocols. After completion of the PCR, amplified DNA were analysed via Gel electrophoresis and gel lanes observed under an illuminator, while determining the sizes of DNA in comparison with a known DNA marker (Thermo Scientific) [21].

Table 1.

Target genes and their respective primers.

Target gene	Primer (Forward and Reverse)	Expected Amplicon Length (bp)	References
pbrA	F-ACTAACGAGGGCATCTGAGC	490	[23]
	R-TGAAACCGTTGCATCACTTGT
cadA	F-TGACTTCCGCGCAATTGTAG	642	[24]
	R-TCGAATGGAATCCGCTACTGA
nreB	F-CCGGAAGAACGCGATTACAC	725	[25]
	R-GCCTTTCGTGGTCTTCTCGT
zntA	F-CGGGTTATTGATGGTCAGCG	289	[26]
	R-AAATGACCGTCAACTGCACC

Screening of MTB for plant growth-promoting properties

To determine the nitrogen-fixing potential of the MTB, each isolate previously grown in nutrient broth is smeared on Burk’s modified nitrogen-free medium containing glucose (1 g), sucrose (1 g), K2HPO₄ (0.06 g), KH₂PO₄ (0.016 g), MgSO₄.7H₂O (0.02 g), NaCl (3 g), CaSO₄.2H₂O (0.005 g), 0.05% Na2MoO₄ (0.5 mL), 0.3% FeSO₄.7H₂O (0.5 mL) and agar (1.5 g) dissolved in distilled water (100 mL). The plates were incubated for 5 days at room temperature. Isolates with distinct growth were considered nitrogen fixers [26].

Phosphate solubilisation was evaluated according to the method described by Alaylar et al. [27], with slight modification (addition of Bromothymol purple) by smearing bacterial colony on National Botanical Research Institute’s Phosphate (NBRIP) agar comprising glucose (1 g), Ca₃(PO₄)2 (0.5 g), MgCl₂6H₂O (0.5 g), KCl (0.02 g), (NH₄)2SO₄ (0.1 g), MgSO₄·7H₂O (0.025 g) and agar (1.5 g), all in 100 mL distilled water and supplemented with 2 drops of Bromothymol purple to assess the extent of solubilisation. Incubation followed for 7 days at 28 °C. The phosphate solubilisation index (PSI) was calculated as reported in equation 1 below;

$$PSI=\frac{\text{Colony diameter}+\text{clear zone diameter}}{\text{colony diameter}}$$ (1)

Potassium solubilisation was determined by smearing bacterial colony on Aleksandrov medium [28] comprising of glucose (0.5 g), MgSO₄ (0.05 g), CaCO₃ (0.01 g), FeCl₃ (0.001 g), potassium alumino silico (0.2 g), agar (2.0 g), all in 100 mL distilled water, the plates were thereafter incubated for 7 days at 30 °C. The extent of solubilisation is calculated as potassium solubilisation index (KSI) as shown in equation 2 below;

$$KSI=\frac{\text{Colony diameter}+\text{clear zone diameter}}{\text{colony diameter}}$$ (2)

Indole-3-acetic acid (IAA) production potential of the isolates was evaluated using the method described by Orhan [26]. Nutrient broth augmented with Tryptophan (0.1 mg/mL) was inoculated with the different isolates, followed by a 2-day incubation at 28 °C on a rotary shaker (150 rpm). Afterwards, the culture was centrifuged at 10000 x g, supernatant was extracted and 0.1 mL of it, 1.5 mL Salkowski’s reagent was added and mixture incubated in the dark for half an hour. A development of pink colour signified IAA production and quantified using a spectrophotometer at 530 nm, optical density generated was compared with a tryptophan standard curve [29] to determine the concentration of IAA produced.

Siderophores production potential of the isolates was evaluated according to the method described by Goswami et al. [30]. Bacterial colony was inoculated on Fiss minimal medium consisting of KH₂PO₄ (0.5 g), glucose (0.5 g), L-asparagine (0.5 g), FeSO₄ (139 μg/L), MgSO₄ (40 mg/L), ZnCl₂ (500 μg/L) and supplemented with 0.5 μM iron. Following a 48-hour incubation at 28 °C, there was centrifugation of the culture at 1000 rpm for 15 minutes, after which 0.5 mL of 2% FeCl₃ was added to the supernatant (0.5 mL). Development of reddish-brown or orange colour signified siderophores production, followed by spectrophotometry to determine the volume of siderophores produced, and subsequently calculated as shown in equation 3 [31] below;

$$\text{Siderophore}(\text{\%})=\frac{\text{Absorbance of reaction sample-Absorbance of control}}{\text{Absorbance of reaction sample}}\times 100$$ (3)

Results

Metal concentrations in the soil samples

Across the three sampling sites, the most abundant contaminant was lead with a concentration of 2596.83 ± 4.48 mg/kg. Notably, amongst all the three sampling sites, Lalupon appeared to be the most polluted, as the test metals were highly concentrated (Lead; 2596.83 ± 4.48 mg/kg, Cadmium; 49.70 ± 0.61 mg/kg, Nickel; 50.05 ± 0.09 mg/kg) and were significantly different (p ≤ 0.05) in comparison with the metals in the other sampling sites, except for zinc that was the most abundant (2160.93 ± 0.37 mg/kg) at sampling site Oke-Omin (Table 2).

Table 2.

Heavy metal concentrations in soils from the different battery waste dumpsites.

Metal	Lalupon	Ile-Igbon	Oke-Omin	CONTROL	WPL
Nickel	50.05 ± 0.09a	23.83 ± 0.15c	33.32 ± 0.57b	18.67 ± 0.09d	50
Cadmium	49.70 ± 0.61a	0.00 ± 0.00d	41.93 ± 0.10b	14.66 ± 0.29c	3
Zinc	1445.07 ± 0.05b	77.93 ± 0.12c	2160.93 ± 0.37a	0.35 ± 0.01d	300
Lead	2596.83 ± 4.48a	1046.00 ± 0.30c	1060.70 ± 0.42b	11.20 ± 0.02d	100

Values are expressed as Means ± Standard deviation of duplicate observations. Means with different superscript alphabets across each row differs significantly from each other at p≤0.05, as determined by One-way Analysis of Variance(ANOVA), with means separated using the Duncan Multiple Range Test; WPL – World Health Organisation Permissible Limit.

Distribution of metal-tolerant bacteria

Table 3 depicts the respective counts for total heterotrophic MTB. A count of 4.48 ± 0.30 LogCFU/mL was obtained from the first sampling at Lalupon, with significant difference (p ≤ 0.05) from the total heterotrophic MTB from sampling sites B and C. At sampling 2, total heterotrophic MTB of 4.30 ± 0.24 LogCFU/mL was counted in soil from Ile-Igbon and showed no significant difference (p > 0.05) in comparison with the total heterotrophic MTB from Lalupon and Oke-Omin. A total number of 132 metal-tolerant bacteria were isolated across the three sampling sites; 34 (25.76 %), 39 (29.55 %) and 59 (44.70 %) were isolated from sampling sites Oke-Omin, Ile-Igbon and Lalupon, respectively (Figure 2).

Table 3.

Total heterotrophic metal-resistant bacterial count (Log CFU/mL) from soil samples.

Sampling Site	A	B
Lalupon	4.48 ± 0.30a	4.05 ± 0.82a
Ile-Igbon	4.40 ± 0.26a	4.30 ± 0.24a
Oke-Omin	4.08 ± 0.43a	3.53 ± 2.35a

Values are Means ± Standard Deviation of duplicate observations. Means with different superscript alphabets down each column differs significantly from each other at p ≤ 0.05 using the New Duncan Multiple Range Test

Figure 2.

Distribution of MTB isolated from the three different sampling locations.

Level of bacterial tolerance to increasing metal concentrations

The tolerance level of the MTB is depicted in Table 4. Amongst the different metals, lead was the most tolerated as all the isolates tolerated it up to 500 μg/mL, whereas the least tolerated metal was cadmium as many of the MTB did not grow at 500 μg/mL. Meanwhile, nickel and zinc were tolerated fairly by a significant number of the isolates. Notably, only 20 (15.15%) of the 132 MTB isolated were multi-metal tolerant, that is, tolerated minimum any three metals up to 400 μg/mL.

Table 4.

Tolerance level (μgmL^-1) of isolates to different test metals.

Isolate	Zinc	Nickel	Lead	Cadmium
ILA2	500	200	500	400
ILA12	500	200	500	500
ILB8	400	100	500	400
ILC4	500	500	500	300
ILC6	500	500	500	400
ILC16	500	500	500	500
ILC17	500	500	500	500
OKA2	500	100	500	400
OKA7	500	100	500	400
OKB9	500	100	500	500
OKB21	500	500	500	300
OKB26	500	500	500	400
LB1	500	400	500	400
LC5	500	100	500	500
LC8	400	200	500	400
LC24	400	100	500	500
LC22	400	300	500	500
LC21	400	500	500	500
OK3	400	300	500	400
OK5	400	400	500	400

Confirmatory identity of selected multi-metal tolerant bacteria

Following 16S rRNA sequencing and BLAST on NCBI, 1 (5%) the 20 multi-metal tolerant bacteria were identified as Serratia marcescens, Bacillus tropicus, Providencia vermicola, Citrobacter freundii and Providencia rettgeri, 2 (10%) as Staphylococcus edaphicus, Mammalicoccus lentus, Alcaligenes ammonioxidans, 3 (30%) as Alcaligenes pakistanensis and 4 (40%) as Pseudomonas aeruginosa (Table 5). Figure 3 shows the phylogenetic associations of the 20 multi-metal tolerant bacteria.

Table 5.

Confirmatory identity of selected multi-metal tolerant bacteria.

S/N	Isolate	NCBI Identity	Similarity %	Accession number
1	ILA2	Pseudomonas aeruginosa	100.00	OR452236
2	ILA12	Bacillus tropicus	99.54	OR452237
3	ILB8	Alcaligenes pakistanensis	99.64	OR452238
4	ILC4	Pseudomonas aeruginosa	99.88	OR452239
5	ILC6	Alcaligenes faecalis	81.44
6	ILC16	Alcaligenes sp.	82.51
7	ILC17	Pseudomonas aeruginosa	100.0	OR452240
8	OKA2	Alcaligenes pakistanensis	83.56
9	OKA7	Mammalicoccus lentus	100.0	OR344100
10	OKB9	Alcaligenes ammonioxydans	93.25	OR452245
11	OKB21	Pseudomonas aeruginosa	99.88	OR452246
12	OKB26	Providencia rettgeri	77.34
13	LB1	Providencia vermicola	98.26	OR344097
14	LC5	Staphylococcus edaphicus	99.25	OR338278
15	LC8	Serratia marcescens	99.6	OR452241
16	LC24	Citrobacter freundii	95.61	OR452243
17	LC22	Staphylococcus edaphicus	100.0	OR344098
18	LC21	Alcaligenes pakistanensis	99.76	OR452242
19	OK3	Mammalicoccus lentus	100.0	OR344099
20	OK5	Alcaligenes ammonioxydans	98.97	OR452244

Figure 3.

Phylogenetic (Neighborhood-Joining) Tree of selected isolates.

Distribution of metal-resistance genes amongst multi-metal tolerant bacteria

Table 6 depicts the distribution of genes coding for resistance to the tested heavy metals amongst the selected multi-metal tolerant bacteria. Of the 20 isolates, 1 (5%) possessed pbrA responsible for resistance to lead, while 5 (25%) possessed cadA responsible for cadmium resistance and 12 (60%) possessed zntA encoding zinc resistance. The gel electrophoresis bands representing the different genes encoding respective metal resistance is shown in Figure 1.

Table 6.

Prevalence of metal resistance-encoding genes in selected bacteria.

Isolate	Genes
	pbrA (490 bp)	zntA (289 bp)	cadA (642 bp)	nreB (725 bp)
Pseudomonas aeruginosa ILA2	-	-	-	-
Bacillus tropicus ILA12	-	-	-	-
Alcaligenes pakistanensis ILB8	-	-	+	-
Pseudomonas aeruginosa ILC4	+	+	-	-
Alcaligenes faecalis ILC6	-	+	+	-
Alcaligenes sp. ILC16	-	+	+	-
Pseudomonas aeruginosa ILC17	-	+	-	-
Alcaligenes pakistanensis OKA2	-	+	-	-
Mammalicoccus lentus OKA7	-	+	-	-
Alcaligenes ammonioxydans OKB9	-	+	+	-
Pseudomonas aeruginosa OKB21	-	+	-	-
Providencia rettgeri OKB26	-	-	-	-
Providencia vermicola LB1	-	+	+	-
Staphylococcus edaphicus LC5	-	-	-	-
Serratia marcescens LC8	-	+	-	-
Citrobacter freundii LC24	-	+	-	-
Staphylococcus edaphicus LC22	-	+	-	-
Alcaligenes pakistanensis LC21	-	-	-	-
Mammalicoccus lentus OK3	-	-	-	-
Alcaligenes ammonioxydans OK5	-	-	-	-

Figure 4.

Gel plates showing bands of selected metal-resistance genes on gel electrophoresis. (a) Upper Well pbrA gene: From Left: DNA Ladder (100bp), Isolate 1-19. Lower wells cadA gene: From left: DNA Ladder (100bp), Isolate 1-19. (b) Upper Well nreB gene: From Left: DNA Ladder (100bp), Isolate 1-19. Lower wells zntA gene: From left: DNA Ladder (100bp), Isolate 1-19.

Plant growth-promoting attributes of selected multi-metal tolerant bacteria

Table 7 shows the varying plant growth-promoting attributes of the selected isolates. Nitrogen fixation activity was exhibited by 15 (75%) of the isolates, while potassium and phosphate solubilisation capability was exhibited by 10 (50%) and 5 (25%), respectively. Meanwhile, Pseudomonas aeruginosa OR452240 exhibited the highest PSI (3.9), while Providencia rettgeri OKB26 had the least PSI. Furthermore, Pseudomonas aeruginosa OR452240 exhibited the highest KSI of 5.7, while least KSI (1.7) was exhibited by Alcaligenes ammonioxydans OR452246.

Table 7.

Plant growth promotion properties of isolates.

Isolate	N2 fixation	PSI	KSI	IAA (μg/mL)	Siderophores (%)
Pseudomonas aeruginosa ILA2	+	3.0	-	25.64	95.26
Bacillus tropicus ILA12	-	-	-	12.18	93.38
Alcaligenes pakistanensis ILB8	+	-	-	22.18	-
Pseudomonas aeruginosa ILC4	+	-	-	-	93.42
Alcaligenes faecalis ILC6	+	-	-	18.91	96.86
Alcaligenes sp. ILC16	-	-	3.8	12.27	-
Pseudomonas aeruginosa ILC17	+	3.9	5.7	21.82	97.58
Alcaligenes pakistanensis OKA2	+	-	-	9.27	80.77
Mammalicoccus lentus OKA7	+	-	4.0	20.82	87.01
Alcaligenes ammonioxydans OKB9	+	-	1.7	-	98.00
Pseudomonas aeruginosa OKB21	+	3.3	-	20.55	98.18
Providencia rettgeri OKB26	+	1.4	3.0	-	96.98
Providencia vermicola LB1	+	2.6	2.9	22.73	97.74
Staphylococcus edaphicus LC5	+	-	-	-	98.20
Serratia marcescens LC8	-	-	-	-	85.5
Citrobacter freundii LC24	-	-	4.3	15.91	95.28
Staphylococcus edaphicus LC22	+	-	4.8	12.55	95.91
Alcaligenes pakistanensis LC21	+	-	-	-	-
Mammalicoccus lentus OK3	+	-	3.2	4.00	89.13
Alcaligenes ammonioxydans OK5	-	-	3.2	-	89.47

N2 fixation: Nitrogen Fixation; PSI: Phosphate Solubilisation Index; KSI: Potassium Solubilisation Index; IAA: Indole-3-Acetic Acid.

Of the 20 multi-metal tolerant isolates, IAA and siderophores were produced by 13 (65%) and 17 (85%), respectively. Amongst these, 98.18% siderophores was produced by Pseudomonas aeruginosa OR452240, whereas 22.83 μg/mL was highest concentration of IAA produced by Providencia vermicola OR344097. However, in comparison with other isolates, the best plant growth-promoting traits were exhibited by Providencia vermicola OR344097, Pseudomonas aeruginosa OR452246 and Pseudomonas aeruginosa OR452240.

Discussion

Of the three sampling sites utilised in this study, Lalupon site was observed to be the most polluted as nickel, cadmium and lead were of elevated concentrations, higher than what was obtainable across the other dumpsites. This observation has been reported by Joseph et al. [16] that Lalupon dumpsite is heavily polluted with these heavy metals. At the different sampling locations, the most abundant contaminant was lead; this observation aligns with the findings from an earlier study on one of the sampling sites, Lalupon, as reported by Joseph et al. [16]. This elevated pollution by lead may be attributed to the consistent deposition of battery wastes from industries on the sites, that result in leaching of toxic metals into the soil. It is noteworthy to highlight that the concentrations of the different tested metals in the soils from the different study area were beyond the limits for soil metal concentrations, as recommended by the World Health Organization [32].

Metal-tolerant bacteria isolated from this study totalled One hundred and thirty-two. Notably, the highest metal-tolerant bacterial number was observed from Lalupon dumpsite and is in alignment with the study of Joseph et al. [16], reporting high number of MTB as well from Lalupon battery waste site. Other authors have reported similar findings from metal-polluted environments including soil and wastewater; Nath et al. [33] isolated 62 MTB from lead- and cadmium-contaminated crop field in Cacchar district, Assam, India. Fashola et al. [34] reported the isolation of 117 distinct culture of MTB from mine tailings in South Africa. Alam et al. [35] reported similar findings in which 198 MTB were isolated from metal-laden effluents from a tannery and receiving soils. This elevated number of MTB isolated from this study is an indication of these bacterial capability to adapt under this environmental stress. Of the 132 MTB, 20 (15.15%) exhibited multi-metal tolerance with 100% tolerance to lead; this aligns with the findings of Khan et al. [36], reporting two isolates showing elevated resistance to lead. Likewise, Nath et al. [33] reported significant tolerance to lead. More so, elevated resistance to lead was exhibited by all MTB isolated from Lalupon dumpsite [16].

In the current study, metal-tolerant bacterial isolates showed significant tolerance to multiple metals and this corroborates reports of Joseph et al. [16] in which 13 (81.25%) of MTB showed resistance to multiple metals. In a study by Fashola et al. [34], 29.91% of 117 bacteria isolated from an abandoned mine in South Africa exhibited resistance to multiple metals. High level of tolerance to multiple metals such as zinc, cobalt, cadmium and lead reaching 1000 mg/kg by bacteria isolated from mine tailings in China has been reported by Yu et al. [37]. This capability to thrive under different environmental stressors including heavy metals may be by using different means such as exclusion, adaptation and other physiological means. Some bacteria may as well utilise efflux pumps, metal-resistance genes and other mobile genetic elements such as plasmids, transposons, etc. [38]. This elevated multiple metal tolerance by these MTB may qualify them as candidates for on-field bioremediation application.

Several bacteria have been implicated in exhibiting resistance to multiple metals. In this present study, the multimetal tolerant bacteria were identified to belong to different genera including Pseudomonas, Alcaligenes, Bacillus, Staphylococcus, Providencia, etc. These groups of bacteria have been recognised as multi-metal resistant bacteria isolated from different metal-polluted environments. In the study by Nath et al. [33], metal-tolerant bacteria isolated from cadmium- and lead-contaminated soils upon identification belonged to species such as Bacillus and Pseudomonas, same as some of the MTB isolated from the current study. Oyewole et al. [39] isolated bacteria of the genera Bacillus, Staphylococcus and Pseudomonas from metal-polluted soils exhibiting high level of metal tolerance. Remarkably, the predominant genera from the current study was Pseudomonas and this is in line with several reports that this species of bacteria have showed remarkable potential of metabolism and tolerance to a broad range of pollutants such as heavy metals, xenobiotics and hydrocarbons [16, 18, 40]; this ability has been attributed to the cell wall architecture of this bacterial species [41].

Many bacteria exhibit tolerance to different pollutants including heavy metals through a number of mechanisms such as physiological and genetic means, which is a necessity for their survival and proliferation, especially under exposure to elevated metal stress and this may be physiological through the use of efflux pumps or genetical, involving metal-resistance genes. A high percentage of MTB from this study harboured genes coding for resistance to zinc and cadmium, while a few to lead. Detection of metal-resistance genes in bacteria isolated from metal-polluted environmental samples has been reported by Adekanmbi et al. [42], in which seven isolates identified as Pseudomonas aeruginosa harboured pbrA encoding resistance to lead were isolated from printery wastewater. Similarly, genes encoding resistance to cadmium, zinc and lead as reported in this study, were detected in 23% of 100 isolated bacteria from soils polluted by heavy metals [43]. The observations of several isolates harbouring metal-resistance genes is an indication that the possible mechanism of resistance to tested metals by these multi-metal tolerant bacteria is genetic using these genes. It is noteworthy to mention that despite elevated resistance being exhibited by the isolates to lead, only 1 isolate harboured the pbrA gene, and this may signify that the elevated concentrations of lead being the most abundant across the sampling sites ensured that the indigenous bacteria developed adaptation mechanisms that are physical or physiological, and not genetical. Meanwhile, the non-detection of nickel-resistance gene nreB in any of the selected multi-metal tolerant bacteria is unsurprising as the selected isolates in the current study did not tolerate elevated concentrations of nickel; earlier studies [16, 34] have attributed this condition to nickel toxicity.

Plant growth-promoting bacteria (PGPB) are ubiquitous and have been reportedly isolated from different sources as beneficial microbes that can serve as alternatives to detrimental compounds embedded in chemical pesticides and fertilizers [44]. In the current study, of 20 selected isolates, 15 (75%) showed nitrogen-fixing capability, an observation corroborating comparatively with 5 isolates with nitrogen-fixing potential reported by AlAli et al. [45]. According to Zhu et al. [46], PGPB are essential in solubilisation of phosphate to release phosphorus from its insoluble state, thus making it accessible for plants. In the current study, as low as 5 bacteria demonstrated phosphate-solubilisation potential, an observation similar to the reports of AlAli et al. [47], in which only 2 metal-tolerant bacteria isolated from a metal-polluted soil solubilized phosphate. Despite the low number of phosphate-solubilizing bacteria from the current study, there is still an indication that these bacteria possess potential to enhance plant growth via release of phosphorus from insoluble phosphate. In terms of potassium solubilisation, only 10 (50%) demonstrated significant potassium solubilisation. A number of authors have reported comparative observations; Verma et al. [48] reported 14 bacteria isolated from soil samples demonstrating significant potassium solubilisation. This elevated number of potassium solubilizers isolated from this study demonstrates their potential for use as biofertilizers, especially in potassium deficient soils as well as those rich in potassium, as they will improve the concentration of soil potassium through enhanced soluble potassium release.

The role of phytohormones such as siderophores and IAA in plant growth cannot be overemphasized, as they help in plant survival when exposed to different environmental stressors such as heavy metals, while also enhancing root development. In the present study, 65% of the isolated produced significant amount of IAA (4.00-22.73μg/mL), similar observation has been reported in an earlier study in which 27 metal-tolerant bacteria isolated from metal-laden soils produced significant amount of isolates [49]. This observation of IAA production by isolates in this study is a criterion qualifying them as candidates for plant growth promotion. Siderophores production enhances the ability of the producing-isolates in inhibiting phytopathogens by sequestering iron, thus reducing its bioavailability and as such these pathogens requiring it for various metabolic processes would be restricted, hence, resulting into their death [50]. In this study, 85% of the multi-metal tolerant bacteria demonstrated siderophores production and this corroborates the findings of Syed et al. [51], in which predominantly Trichioderma sp. and Pseudomonas sp. demonstrated significant siderophores production.

Notably, many isolates from this study demonstrated dual ability of multi-metal tolerance and multiple PGP attributes including nitrogen fixation, potassium- and phosphate solubilisation alongside IAA and siderophores production. This dual ability was reported in bacteria isolated from soils polluted with lead and zinc in Turkey, exhibiting both metal-tolerance and PGP characteristics [52]. From the 20 isolates, Providencia vermicola and Pseudomonas aeruginosa demonstrated the best multi-metal tolerance and PGP properties. These species of bacteria have been previously reported to demonstrate PGP features and tolerance to multiple metals at elevated concentrations [51, 53].

Conclusions

From the findings of this study, it is evident that the continuous release of battery wastes into the environment significantly leaches toxic metals into the soil which may be detrimental to the health of the soil. Furthermore, this study is an indication that these metal-polluted sites can enhance metal tolerance in soil bacteria. The selected metal-tolerant bacteria from this study harboured different metal-resistance genes that may have enhanced their elevated metal tolerance. More so, this study has indicated that plant growth-promoting bacteria can be present anywhere, metal-polluted soils not excluded. Furthermore, Providencia vermicola OR344097, Pseudomonas aeruginosa OR452240 and Pseudomonas aeruginosa OR452246 demonstrated the best multi-metal tolerance and plant growth-promoting traits, thus are good candidates for application in on-site enhancement of plant growth.