Introduction

Pakistan is one of the countries which is facing severe problem of water shortage. Due to scarcity of good quality water, farmers are using wastewater for irrigation purposes (Agrawal et al. 2014; Lam et al. 2015) around metropolitans that not only compensates for the water shortage but also provides essential plant nutrients to crops (Agrawal et al. 2014; Nafchi 2017). It, however, contains different kinds of organic and inorganic pollutants (Chibuike and Obiora 2014; Ahmad et al. 2018a). Heavy metals are among the most important inorganic pollutants (Ahmad et al. 2018a) which are incorporated into the sewage systems through various sources including industries, farms wastes, agrochemicals and automobiles (Akpor et al. 2014; Ferronato and Torretta 2019). Toxic heavy metals accumulate in arable soils and move into food chain, thus disturbing the natural ecosystem. These toxic metals pose serious threat to food security and health by deteriorating the quality of produce (Iqbal et al. 2016; Ojuederie and Babalola 2017), particularly in developing countries.

Heavy metals present in wastewater act as toxicants causing serious challenges for the aquatic life (Gheorghe et al. 2017) and pose a serious threat to public health (Naik et al. 2012). In plants, heavy metals hinder the normal metabolism by disturbing physiological and biochemical process (Ackova 2018). Lead, a non-essential heavy metal, is highly toxic to plants, animals, human and even microbes (Nas and Ali 2018). It is produced as a byproduct of several processes such as burning of fossil fuels, ore processing, mining (Murthy et al. 2014). Moreover, Pb contamination in wastewater through paint production and usage, plumbing materials, batteries and agrochemicals, etc. is a serious concern for vegetables growing around cities (NASEM 2017). Concentration of this highly toxic metal is already more than the prescribed WHO levels in different areas of Pakistan (Rasheedet al. 2014). There are several conventional and physical methods to remove metals from the environment, but these are expensive and not that much effective in case of lower concentrations. In such cases, utilizing microbes with ability to stabilize these heavy metals can be a good strategy for growing vegetables with wastewater. Although, the heavy metal tolerant microbes are successfully being used for bioremediation/decontamination of heavy metal contaminated soils (Ahmad et al. 2018a), they can also stabilize heavy metals in soils thus limiting their accumulation in above ground parts of plants (Saran et al. 2020). It is not only a cheap and efficient method but also an eco-friendly approach that can help to utilize wastewater. Lead tolerant microorganisms belonging to different genera i.e., Micrococcus, Staphylococcus, Lysinibacillus Escherichia, Flavobacterium, Bacillus, Klebsiella, Eenterobacter, Shigella, Salmonella, Enteroccocus and Gemella have been isolated from wastewater, plant rhizosphere and sewage sludge in previous studies (Saleem et al. 2015; Benmalek and Fardeau 2016; Marzan et al. 2017; Kamaruzzaman et al. 2020). The plasma membrane of these bacteria possesses the ability to reduce the entry of such heavy metals in the cytoplasm. Owing to this unique character, these microbes can grow and survive even in such adverse environmental conditions where, otherwise life is impossible (Wang and Chen 2009). The important mechanisms adopted by these microbes for metal detoxification include adsorption by extracellular polysaccharides, absorption, precipitation, complexation and ion exchange (Jaroslawiecka and Piotrowska-Seget 2014). Phytostabilization of heavy metals can be helpful to decrease their toxicity due to reduced uptake and accumulation in crop plants (Saengwilai et al. 2019).

In this context, present study was conducted to isolate, characterize and identification of Pb-tolerant bacterial strains from wastewater to be used as inoculants for growing vegetables by utilizing wastewater as irrigation source.

Materials and Methods

Isolation of heavy metal tolerant bacteria

Lead tolerant bacteria were isolated by using Luria Bertani plates amended with Pb(NO₃)₂ (700 mg L^-1 of lead). The serial dilutions of wastewater samples were prepared and then the wastewater dilutions were inoculated on agar plates and incubated at 37°C for 48 h. Bacterial colonies differing in appearance were selected and purified (Marzan et al. 2017). The pure cultures were preserved at 4°C for experimentation.

Tube dilution method

Lead resistance of isolates was also checked through tube dilution method. The isolates were inoculated on nutrient broth, containing 1000 mg/mL^-1 lead as Pb(NO₃)₂ in 25 mL test tubes. The cultures were incubated at 28°C (ideal temperature for bacterial growth in laboratory) on shaking incubator for 3 days. The growth (optical density) of the isolates was measured on spectrophotometer (Cary 60, Agilent, USA) at 600 nm wavelength. Experiment was done in triplicate and repeated to confirm the results. The bacterial isolates with the highest optical density were selected for further studies (Neethu et al. 2015).

Exopolysaccharides production

Bioassay was performed to study the exopolysaccharides production ability of isolates by following the method of Tallgren et al. (1999). Exopolysaccharides produced by bacteria were studies because it makes complexes with heavy metals thus reduce their uptake by crop plants. These Pb-tolerant strains with ability to produce exopolysaccharide not only improved spinach growth but also decreased the Pb uptake by stabilizing it in the root zone. On the other hand, siderophore production was determined because bacteria produced some iron-chelating agents which facilitate availability of iron to plants. The strains were incubated on ATCC No. 14 medium for seven days at 28°C temperature. Bacterial colonies which were able to form thick slime (mucoid) around colonies were taken as positive for exopolysaccharides production.

Siderophores production

Siderophores production by the bacterial isolates was determined by universal Chrome Azurol Sulphonate (CAS) assay (Schwyn and Neilands 1987). For this purpose, CAS reagent was prepared as described by Schwyn and Neilands (1987) and inoculated with respective bacterial strains. The development of orange zone around the colonies was observed as positive for siderophores production.

Jar trial

Based on in vitro characterization i.e., lead resistance, exopolysaccharides production and siderophore production, the Pb-tolerant isolates were selected and were evaluated for their effectiveness to improve growth of spinach seedlings under axenic conditions. The bacterial culture was prepared by growing bacterial strains separately in 100 mL broth following by incubation at 28°C for 2 days. The spinach seeds were disinfected with ethanol (95%) and HgCl₂ (0.2%) solution for 3 min and inoculated with respective isolates by dipping in broth for ten minutes. Four levels of lead (0, 300, 600 and 900 mg Pb kg^-1 of sand) were prepared in sand by artificially contaminating using lead nitrate salt as source of Pb. Jars were filled with sand, moistened with water and sterilized in autoclave. Three extra jars of each treatment were maintained and analyzed at two times during the experiment to ensure that Pb should not leach down from the root zone. For control, the seeds were treated with sterilized broth. Inoculated seeds were sown in autoclaved glass jars filled with sand having different levels of lead as described above. Jars were placed in growth room at 28 ± 1ºC adjusted to 10 h light and 14 h dark period. The jars were arranged in Completely Randomized Design (CRD) with three replications for each treatment. Sterilized Hoagland solution was applied as source of nutrients to growing seedlings. After 30 days of sowing, data regarding growth i.e. root/shoot parameters were recorded.

Lead analysis in spinach seedlings

After harvesting, spinach seedling was air dried and then oven dried in oven at suitable temperature. Ground the oven dried sample in pestle and mortar, digest the sample and analyzed lead (Pb) concentration using Atomic Absorption Spectrophotometer (Model; AAS 240 FS, Agilent, U.S.A.).

Characterization of plant growth promoting bacteria

The cell shape and Gram staining test of bacteria was carried out after 48 h of growth on agar plates by following the method of Vincent (1970).

Bacterial isolates were tested for their ability to solubilize inorganic zinc source (ZnO). Twenty-four-hour old bacterial colonies were inoculated in the center of Pikovskaya agar plates and incubated at 28 ± 1°C for seven days. Development of clear zone around the colony was taken as positive for zinc solubilization (Pikovskaya 1948). For phosphate solubilization, agar plates were prepared with tricalcium phosphate as source of phosphorus and inoculated with respective bacterial isolates. The plates were incubated for seven days at 28 ± 2°C. Development of clear zone around the colonies was taken as positive result of phosphate solubilization (Goldstein 1986). Hydrogen cyanide (HCN) production was determined as described by Lorck (1948). For NH₃ production assay, bacterial colonies were inoculated in peptone water and incubated for 72 h 28 ± 2°C. The development of brown to yellow color was noted as positive result for NH₃ production (Dye 1962). The indole acetic acid production (IAA) was determined by following the standard protocol of Loper and Schroth (1986). The nutrient broth was prepared with L-tryptophan and inoculated with different isolates. These were incubated at 28 ± 2°C for seven days. After seven days, broth was centrifuged and the resulted supernatant was mixed with orthophosphoric acid and Salkowski reagent. The formation of pink color was taken as positive for IAA production. The optical density (OD) values were recorded at OD₅₃₀ and IAA level was determined by standard IAA graph.

Determination of minimum inhibitory concentration (MIC)

Minimum inhibitory concentration was determined by the plate dilution method against lead, as Pb(NO₃)₂ by gradually increasing the concentration of the heavy metal on LB medium until the strains failed to develop colonies on the plates (Vela-Cano et al. 2014). The starting concentration of Pb was 1000 mg L^-1 and the isolates growing on lower concentration were transferred to higher concentration on LB agar plates.

Bacterial growth EPS production (quantitative) by PGPR strains under Pb stress

The selected PGPR strains with ability to improve spinach growth under Pb stress were grown at different concentrations of Pb i.e., 0.8, 1.6, 2.4, 3.2 g L^-1 in Luria Bertani broth to quantify the effect of Pb on their growth, and EPS production ability. Three tubes were prepared and maintained for each concentration. The liquid culture tubes were incubated at 32ºC in shaking incubator at 100 rpm. The optical density (OD₆₀₀) of cultures was measured using UV spectrophotometer (Model Cary 60, Agilent, USA) after 48 h as described by Raja et al. (2006). The EPS production by PGPR strains was also estimated after 48 h using spectrophotometer according to the method of Dubois et al. (1956).

Lead removal by bacterial strains

Bacterial isolates were grown in 50 mL Erlenmeyer flasks containing LB broth medium and placed on rotary shaker for one hour at 150 rpm. Then, 100 mg L^-1 of sterilized Pb as lead nitrate was added separately in every culture flask and again incubated for 24 h under same conditions. In order to determine the amount of Pb retained, the bacterial culture was centrifuged at 5000 rpm for 15 min. The supernatants were separated, digested with concentrated HNO_3, filtered and analyzed by Atomic Absorption Spectrophotometer (Model AAS 240 FS, Agilent, U.S.A.) as described by Vela-Cano et al. (2014). Each treatment was comprised of three replicates and the analyses were repeated to confirm the results.

Identification of bacterial strains through 16S rRNA sequencing

The Pb-tolerant isolates showing better results in jar trial were identified by using 16S rRNA sequencing by following the method as described by Hussain et al. (2011). The resulted partial sequences were analyzed using Blastn analysis option on NCBI website. Sixteen closely related nucleotide sequences were aligned using muscle alignment option in MEGA7 software (Kumar et al. 2016). The evolutionary history was inferred by Neighbor-Joining method (Saitou and Nei 1987) and evolutionary distances were computed by using the maximum composite likelihood method (Tamura et al. 2004).

Statistical analysis

Experiments conducted in jar trial data were statistically analyzed using complete randomized design (CRD), means were compared by using LSD, and Excel (MS office 2010) for respective tests, where applicable, for the purpose to compare the treatments means (Steel et al. 1997).

Results

Isolation of heavy metal tolerant bacteria

Out of forty bacterial isolates, twenty-two showed lead resistance in plate assay as they were able to grow on lead nitrate amended Luria Bertani media (700 mg L^-1 Pb) and were further confirmed in broth assay. Twenty isolates showed exopolysaccharides production, while 15 isolates were positive for siderophore production. Among these, 10 isolates were selected which exhibited the maximum growth (OD₆₀₀) in lead amended nutrient broth, (1000 mg L^-1 of Pb) and were positive for exopolysaccharides and siderophores production (Table 1). The results of the broth assay (Fig. 1) showed that the maximum optical density was observed in the case of N11 followed by N35 and N18.

Table 1: Isolation of lead resistant bacteria and their selection based on special tests

Isolates	Characterization of bacteria
	Lead resistant	Exopolysaccharides	Siderophores
	(700 mg L^-1 Pb)	(Plate assay)	(Plate assay)
N1	+	+	-
N2	-	+	-
N3	+	-	-
N4	-	-	++
N5	-	+	-
N6	-	+	-
N7	++	+++	++
N8	+++	+++	+++
N9	+	-	-
N10	-	+	-
N11	+++	+++	+++
N12	++	-	-
N13	-	-	-
N14	+	-	-
N15	-	-	+
N16	-	+
N17	+	+	-
N18	+++	+++	+++
N19	+	-	-
N20	-	+	-
N21	-	-	-
N22	-	-	-
N23	+++	++	++
N24	+	-	-
N25	++	+++	+++
N26	-	-	-
N27	+	-	-
N28	-	-	-
N29	+++	+++	+++
N30	-	+	++
N31	-	-	-
N32	+	-	+
N33	-	-	-
N34	+	-	+
N35	+++	+++	++
N36	-	-	-
N37	+	+	-
N38	-	-	-
N39	++	+++	++
N40	++	++	+++
(+++) = (++) = (+) = growth, (-) = no growth

Fig. 1: Growth pattern of isolates in Luria Bertani medium supplemented with 1000 mg L^-1 of Pb

Effectiveness of lead tolerant bacteria under axenic conditions

These isolates were screened for their ability to improve growth of spinach seedlings in jar trial using lead nitrate [Pb(NO₃)₂] as source of lead at different levels (0, 300, 600, and 900 mg kg^-1). These isolates promoted the spinach growth parameters in jar trial under axenic conditions but with variable response. Shoot length of spinach was significantly decreased in lead contamination that was improved by the Pb Tolerant plant growth promoting bacterial strains in lead contaminated soil (Table 2). The results showed that inoculation of Pb Tolerant bacterial strain N11 increased the shoot length (up to 29.82%) at 900 mg kg^-1 as compared to respective control. Results regarding shoot dry weight revealed that Pb contamination also reduced shoot dry weight with increasing concentration at all levels (Table 2). The most severe reduction was recorded at the highest level (900 mg kg^-1 of Pb). Inoculation with Pb Tolerant bacterial strains significantly increased shoot dry weight of plants at all levels of lead contamination. At highest concentration of lead (900 mg kg^-1 of Pb), the maximum improvement in dry weight of spinach plant (28.30%) as compared to respective control was observed by the Pb tolerant bacterial strain N11.

Table 2: Effect of Pb-tolerant plant growth promoting rhizobacteria on shoot length and shoot dry weight of spinach plants under Pb-stressed axenic conditions in jar trial (n = 3)

Isolate	Lead levels (mg kg^-1)
	0	300	600	900
	Shoot length (cm)
Control	14.15 ± 0.5 j-o	12.98 ± 0.2 l-p	11.22 ± 0.4 r-v	9.94 ± 0.2 v
N7	15.29 ± 0.3 d-j	14.94 ± 0.2 e-j	12.17 ± 0.3 p-t	10.47 ± 0.2 uv
N8	16.95 ± 0.2 a-c	16.55 ± 0.3 a-e	14.13 ± 0.3 j-o	12.71 ± 0.2 o-r
N11	18.05 ± 0.6 a	16.67 ± 0.4 a-d	14.55 ± 0.2 h-l	12.90 ± 0.4 m-q
N18	17.98 ± 0.1 ab	16.62 ± 0.3 a-d	14.40 ± 0.3 i-n	12.81 ± 0.5 n-r
N23	16.07 ± 0.3 c-h	14.81 ± 0.5 f-k	12.80 ± 0.5 n-r	11.00 ± 0.4 s-v
N25	16.43 ± 0.4 a-f	15.97 ± 0.4 c-i	12.87 ± 0.2 m-r	12.58 ± 0.4 o-s
N29	18.04 ± 0.3 a	16.37 ± 0.3 b-f	14.41 ± 0.3 i-n	12.71 ± 0.4 o-r
N35	16.64 ± 0.3a-d	15.91 ± 0.2 c-i	13.25 ± 0.1 k-p	12.01 ± 0.4 p-u
N39	15.13 ± 0.3 d-j	14.65 ± 0.2 g-k	12.67 ± 0.2 o-r	11.30 ± 0.3 q-v
N40	16.25 ± 0.3 c-g	14.47 ± 0.2 h-m	12.17 ± 0.6 p-t	10.68 ± 0.5 t-v
LSD (P ≤ 0.05)	1.6453
	Shoot dry weight (g plant^-1)
Control	0.56 ± 0.01 fg	0.50 ± 0.01g-j	0.44 ± 0.01 i-n	0.35 ± 0.01 o
N7	0.60 ± 0.01 c-f	0.58 ± 0.01 d-f	0.47 ± 0.01 i-m	0.38 ± 0.01 no
N8	0.71 ± 0.01ab	0.64 ± 0.01 c-e	0.56 ± 0.01f-h	0.45 ± 0.01 i-n
N11	0.72 ± 0.02 a	0.65 ± 0.02 b-d	0.57 ± 0.01 e-g	0.45 ± 0.01 i-m
N18	0.71 ± 0.01 ab	0.64 ± 0.01 c-e	0.56 ± 0.02 f-h	0.45 ± 0.02 i-n
N23	0.62 ± 0.02 c-f	0.57 ± 0.02 e-g	0.50 ± 0.02 g-j	0.38 ± 0.01 no
N25	0.67 ± 0.01 a-c	0.62 ± 0.01c-f	0.51 ± 0.02 g-i	0.42 ± 0.02 k-o
N29	0.71 ±0.01ab	0.64 ± 0.02 c-e	0.56 ± 0.01 f-h	0.45 ± 0.01 i-n
N35	0.66 ± 0.01 a-c	0.61 ± 0.01 c-f	0.50 ± 0.03 g-j	0.43 ± 0.01 j-n
N39	0.66 ± 0.03 a-c	0.56 ± 0.02 fg	0.49 ± 0.01 h-k	0.41 ± 0.01 l-o
N40	0.62 ± 0.01 c-f	0.56 ± 0.01f-h	0.48 ± 0.02 i-l	0.39 ± 0.01 m-o
LSD (P≤0.05)	0.0704

Means sharing different letters are statistically significant from each other at 5% level of probability.

Table 3: Effect of Pb-tolerant plant growth promoting rhizobacteria on root dry weight of spinach plants under Pb-stressed axenic conditions in jar trial (n = 3)

Isolate	Lead levels (mg kg^-1)
	0	300	600	900
	Root dry weight (g plant^-1)
Control	0.41 ± 0.02 d-h	0.35 ± 0.01 k-p	0.29 ± 0.01 q-s	0.25 ± 0.01 s
N7	0.45 ± 0.01 b-e	0.40 ± 0.01 e-k	0.32 ± 0.01 n-r	0.28 ± 0.02 rs
N8	0.51 ± 0.01 ab	0.45 ± 0.02 c-f	0.36 ± 0.01 i-o	0.32 ± 0.01 n-r
N11	0.52 ± 0.01 a	0.44 ± 0.01 c-g	0.37 ± 0.003 h-n	0.32±0.00 m-r
N18	0.53 ± 0.001 a	0.45 ± 0.01 c-f	0.37 ± 0.002 h-m	0.31 ± 0.01 o-r
N23	0.46 ± 0.02 b-e	0.39 ± 0.02 g-k	0.32 ± 0.01 m-r	0.28 ± 0.01 r-s
N25	0.48 ± 0.01 a-c	0.41 ± 0.02 e-j	0.33 ± 0.01 l-q	0.31 ± 0.01 o-r
N29	0.53 ± 0.01 a	0.45 ± 0.01 b-e	0.38 ± 0.002 h-l	0.33 ± 0.01 l-r
N35	0.49 ± 0.01 a-c	0.41 ± 0.01 d-i	0.35 ± 0.004 j-o	0.30 ±0.01 p-s
N39	0.47 ± 0.02 b-e	0.40 ± 0.01 f-k	0.32 ± 0.01 l-r	0.28 ± 0.01 r-s
N40	0.46 ± 0.01 b-d	0.40 ± 0.001 f-k	0.31 ± 0.01 o-s	0.29 ±0.00 q-s
LSD (P≤0.05)	0.0553

Means sharing different letters are statistically significant from each other at 5% level of probability

Soil treated with lead also reduced the root dry weight of spinach seedlings as compared to control plants (Table 3). It was observed that application of Pb Tolerant bacterial strains decreased the toxic effects of lead on root dry weight and improved the root dry weight at all levels of lead stress as compared to control plants. Maximum improvement (26.31%) in root dry weight as compared to respective control plants was found by the inoculation with Pb Tolerant bacterial strain N11 at 900 mg kg ^-1 of Pb.

Lead analysis in spinach seedlings

Results regarding lead contents in roots (Table 4) showed that exposure to lead particularly at higher levels increased accumulation of Pb in roots. The Pb accumulation in spinach roots increased by increasing metal concentration and the maximum accumulation of lead was observed at 900 mg of Pb. However, inoculation with Pb Tolerant bacterial strains N11 and N18 decreased metal contents upto18% at the highest levels of Pb stress (900 mg kg^-1). The data regarding shoot lead contents also showed that lead contents increased with increasing metal stress. However, the metal uptake in shoot was lower as compared to roots of spinach plants. It was observed that inoculation of Pb tolerant bacterial strain N11 decreased the lead contents in shoot up to 19% at 900 mg kg^-1 Pb as compared to respective control plants (Table 4).

Table 4: Effect of Pb-tolerant plant growth promoting rhizobacteria on Pb contents roots and shoots of spinach plants under Pb-stressed axenic conditions in jar trial (n = 3)

Isolate	Lead levels (mg kg^-1)
	0	300	600	900
	Root lead contents (mg kg^-1)
Control	1.20 ± 0.07 w	21.1 ± 1.3 q	37.3 ± 1.0 i	59.7 ± 1.1 a
N7	1.10 ± 0.06 w	20.8 ± 0.9q	33.7 ± 1.0 k	58.1 ± 0.8 b
N8	0.94 ± 0.05 w	17.2 ± 1.0 uv	31.8 ± 0.8 l	52.1 ± 0.7 f
N11	0.90 ± 0.06 w	16.8 ± 0.5 v	27.8 ± 0.7 p	48.2 ± 1.4 h
N18	0.95 ± 0.08 w	17.8 ± 0.7 t	30.6 ± 1.4 l	48.7 ± 1.2 h
N23	1.04 ± 0.06 w	18.3 ± 1.2 s	33.5 ± 1.7 k	53.9 ± 1.4 d
N25	0.99 ± 0.02 w	17.6 ± 0.3 tu	32.0 ± 0.5 l	52.8 ± 1.5 e
N29	0.95 ± 0.05 w	17.5 ± 0.7 tu	29.6 ± 0.9 n	49.5 ± 1.5 g
N35	1.00 ± 0.08w	16.8 ± 1.2 v	28.7 ± 0.9 o	52.5 ± 1.7 ef
N39	1.11 ± 0.07 w	17.5 ± 0.8 tu	33.7 ± 0.8 jk	55.1 ± 1.4 c
N40	1.18 ± 0.05 w	19.6 ± 0.9 r	34.2 ± 0.9 j	55.1 ± 1.0 c
LSD (P ≤ 0.05)	0.4754
	Shoot lead contents (mg kg^-1)
Control	0.77 ± 0.07 w	9.64 ± 1.8 p	17.50 ± 1.6 g	23.67 ± 1.2 a
N7	0.72 ± 0.01 w	9.20 ± 0.7 pq	16.50 ± 0.7 hi	23.23 ± 1.7 a
N8	0.62 ± 0.01 w	8.61 ± 1.7 r-t	15.00 ± 0.9 lm	20.27 ± 1.2 d
N11	0.59 ±0.02 w	7.55 ± 1.5 v	14.30 ± 1 n	18.87 ± 1.2 f
N18	0.66 ± 0.02 w	7.85 ± 0.3 uv	13.80 ± 0.9 o	19.37 ± 1.1 e
N23	0.71 ± 0.02 w	9.06 ± 0.7 qr	16.67 ± 1.4 hi	22.23 ± 0.8 b
N25	0.69 ± 0.01 w	8.58 ± 1.6 r-t	15.73 ± 0.7 jk	21.47 ±0.5 c
N29	0.58 ± 0.02 w	8.15 ± 0.9 tu	14.77 ± 1.1 mn	20.63 ± 1.1 d
N35	0.65 ± 0.05 w	8.41 ± 1.5 st	15.33 ± 0.8 kl	21.40 ± 0.3 c
N39	0.73 ± 0.01 w	8.87 ± 0.8 q-s	16.67 ± 1.1 h	22.40 ± 0.3 b
N40	0.75 ± 0.04 w	9.12 ± 2.6q	16.07 ± 1 ij	23.30 ± 0.9 a
LSD (P ≤ 0.05)	0.4952

Means sharing different letters are statistically significant from each other at 5% level of probability.

Characterization of plant growth promoting bacteria

Morphological and plant growth promoting characteristics of selected isolates are given in Table 5. The results regarding morphological characteristics exhibited that isolates have different colony color, shape, margin with various appearance however belong to Gram positive nature. In zinc solubilization test, all selected isolates N8, N11, N18, N25, N29, and N35 showed zinc solubilization potential by producing a clear zone of various diameters in plate assay. Two isolates namely N8 and N11 showed positive result in phosphate solubilization test. Five isolates N8, N11, N18, N29, and N35 indicated HCN production ability. All six isolates were found to be positive in ammonia production. All tested isolates appeared brown in color on lead incorporated medium. The results obtained from MIC analysis revealed that N11 was the most Tolerant bacteria followed by N18, N8, N29, N25 and N35. The minimum inhibitory concentration of Pb-tolerant bacteria was found up to 3200, 3100, 2700, 2400, 2350 and 2300 mg L^-1 as shown in Table 6.

Table 5: Morphological, biochemical and plant growth promoting characteristics of selected isolates

Bioassays/ morphological traits	Bacteria isolates
	N8	N11	N18	N25	N29	N35
Color	White	yellow	white	white	white	pink
Shape	Round	irregular	irregular	irregular	round	irregular
Size	Medium	small	medium	large	large	large
Margin	Entire	undulate	entire	entire	entire	undulate
Elevation	Raised	raised	raised	flat	raised	flat
Appearance	Smooth	smooth	smooth	smooth	smooth	rough
Gram staining	+ve	+ve	+ve	+ve	+ve	+ve
PGPR traits
Zinc solubilization	+++	+++	+++	+	+++	++
Phosphate solubilization	+++	+	-		-
HCN production	++	++	+++	-	+	+++
Ammonia production	+++	+	+++	++	++	+

(+++) = (++) = (+) = growth, (-) no growth

Table 6: Minimum inhibitory concentration (mg L^-1) of Pb as Pb(NO₃)₂ for selected isolates and IAA production by these bacteria (n =3)

Isolate	Minimum inhibitory conc.	IAA production (µg mL^-1)
	Pb^2+ (mg L^-1)	Without Tryptophan	with Tryptophan
N8	2700	0.8 ± 0.09	8.2 ± 0.12
N11	3200	1.8 ± 0.05	8.6 ± 0.09
N18	3100	6.5 ± 0.11	23.6 ± 1.15
N25	2350	0.8 ± 0.07	8.9 ± 0.43
N29	2400	1.7 ± 0.09	10.2 ± 0.65
N35	2300	0.5 ± 0.04	8.5 ± 0.54

All selected isolates produced IAA but the maximum IAA production (23.6 µg mL^-1) was observed by the strain N18. It is summarized that isolates N8, N11 and N18 possess maximum plant growth promoting characteristics. The effect of lead on bacterial growth at different levels (Fig. 2) showed a decrease in bacterial growth with increasing metal concentration. The maximum growth was observed at control having no metal concentration. Most isolates were tolerant to lead at average concentration (1.6g of Pb). The maximum optical density was given by N11 when compared with other strains at different levels. The data regarding EPS production by PGPR strains (Fig. 3) showed that these strains significantly varied in their ability to produce EPS under Pb stress. Maximum EPS production (133.54 µg mL⁻¹) was observed by N11 at 0.8 g L^-1 of Pb stress. The EPS production by all strains was decreased with increasing level of Pb stress but N11 has significantly higher EPS production at all levels of metal stress. The result regarding metal removal efficiency (Fig. 4) revealed that all strains were able to remove lead. The maximum removal capacity was exhibited by N18 (72%) followed by N11 (69%). The N29, N35 were also able to remove Pb in the range of 56 and 51%, respectively, while N25 was able to remove lead by only 44%.

Fig. 2: Effect of Pb stress on growth of PGPR strains in broth culture (n =3); Means sharing different letters are statistically significant from each other at 5% level of probability

Fig. 3: Effect of Pb stress on exopolysaccharides (µg mL^-1) production of PGPR strains in broth culture (n =3); Means sharing different letters are statistically significant from each other at 5% level of probability

Fig. 4: Lead removal / biosorption by PGPR strains in LB medium under lead stress; (n =3); Means sharing different letters are statistically significant from each other at 5% level of probability

Identification of selected isolates

The selected isolates were identified based on 16S rRNA partial gene sequencing. The phylogenetic tree showed that all the identified strains have evolutionary relationship with genus Bacillus. Based on similarity as inferred during phylogenetic analysis, these strains were identified and are submitted to database of National Center for Biotechnology Information (NCBI; website: https://www.ncbi.nlm.nih.gov/) under accession numbers as B. megaterium strain N8 (MK999909), B. safensis strain N11 (MK999910), B. paranthracis strain N18 (MK999911), B. velezensis strain N25 (MK999912), B. megaterium strain N29 (MK999913) and B. subtilis strain N35 (MK999914) respectively.

Discussion

In present study out of 40 bacterial isolates, 22 bacterial isolates were tolerant to Pb that might be due to production of exopolysaccharides and siderophores by these strains as observed during in vitro studies. Previously, siderophores production by metal-resistant bacterial strains has been reported (Rajkumar et al. 2010). In our study, the strains that were positive in siderophores and exopolysaccharides production. Metal tolerant microorganisms that can produce siderophores are effective for growth and survival of plants under metal polluted environment as siderophores produced by these microbes not only chelate iron but also other metals (Rajkumar et al. 2010). The complexed metals are less toxic due to inactivation through complexation with chelating substances produced by microbes (Gao et al. 2010). Previous studies also report the production of extracellular polymers by microbes and possible protection of cellular components through attachment with metal cations (Bruins et al. 2000). On the other hand, exopolysaccharides producing (EPS) bacterial strains can be helpful in establishing biofilms formation under metal stress which leads to enhanced adsorption and stabilization of these metals in soil (Marchal et al. 2011).

Results of the present study showed that Pb application decreased the spinach growth as compared to plants unstressed control plants. Decrease in plant growth due to presence of excess heavy metals in growth medium is a common feature as reported in previous studies in different plant species (Tangahu et al. 2011; Abdelkrim et al. 2018; Naveed et al. 2020). Lead is toxic to plants that disturbs the physiological processes and antioxidant enzyme systems (Arif et al. 2019). Reduction in plant growth by Pb might also be because lead decreased photosynthetic activity, reduced mineral nutrition, disturbed membrane structure and its permeability, changed hormonal status by altering water balance of plants (Nas and Ali 2018). Inoculation with Pb-tolerant PGPR in Pb amended soil has been reported to decrease the negative effects of Pb thus improved plant growth in Pb contaminated soil. The similar results due to inoculation with Pb tolerant PGPR have also been reported in other studies (Souhir et al. 2018; Abdelkrim et al. 2020) where they attributed the improvement in plant growth with phosphatase activity (Ghoreishi and Etemadifar 2017; Dalyan et al. 2019), siderophore production (Grobelak and Hiller 2017) and development of systematic resistance in plants against abiotic stresses. It has been observed that inoculation with Pb tolerant B. subtilis (PbRB3) regulated the Pb toxicity induced negative effects on mung bean plants (Arif et al. 2019). Similarly, in another study, separate and combined inoculation with Pb-tolerant PGPR strains B. paralicheniformis YSP151 and Brevibacterium frigoritolerans YSP40 enhanced the growth of B. juncea plants grown under metal-stressed conditions (Yahaghi et al. 2018).

Results of our studies showed that increased concentration of Pb in root zone enhanced the uptake of Pb by spinach plants growing under axenic conditions however inoculation with Pb-tolerant bacterial strains reduced Pb uptake by plant roots and shoots as compared to uninoculated stressed plants. Maximum decrease in Pb uptake by spinach roots and shoots was observed in plants inoculated with strain N11 that might be due to higher production of exopolysaccharides by N11 as compared to other strains as observed under our studies. The reduction in metal uptake by inoculated plants has also been reported by Pramanik et al. (2017) where they reported that K5 strain positively affected the plant growth by lowering the metal uptake. They attributed the decrease in metal uptake with production of EPS by bacterial strain. The EPS produced by bacterial strain helped in the sequestration of metals thus decreased the uptake by crop plants. Similarly, Maity et al. (2019) reported that inoculation with B. subtilis C (225) effectively immobilized the heavy metal in soil by mineral precipitation. They reported that inoculation helped in transforming heavy metals from bioavailable fraction to residual fraction thus lowering their uptake by crop plants. It was also observed that Pb contents were more in roots as compared to shoots of spinach plants that might be due to precipitation of Pb in soil and sequestration of up taken Pb into roots due to inoculation with Pb tolerant PGPR which ultimately decreased the its translocation into edible portion (Wani and Khan 2010). Thus, microbial induced mineral precipitation (MIMP) can be used to stabilize heavy metals as an effective strategy for growing leafy vegetables under metal contaminated soils / wastewater irrigated soils. There are certain bacterial species which have been proved effective to reduce metal toxicity through different mechanisms such as solubilization and stabilization (Ojuederie and Babalola 2017; Hamidpour et al. 2019). Stabilization of these metals in soils irrigated with wastewater can reduce their uptake by vegetables (Saran et al. 2020).

In our studies, the Pb tolerant PGPR strains were characterized for different morphological, biochemical, plant growth promoting characteristics. It was observed that these strains are positive in more than one characteristic, but they varied in different attributes.

Results of our studies showed that Pb-tolerant bacterial strains have multiple PGP traits including Zn solubilization, phosphate solubilization, ammonia production, HCN production and IAA production. The strains belonging to genus Bacillus have been documented to possess multiple PGP traits (Mumtaz et al. 2017; Ahmad et al. 2018b). These strains can improve the growth and nutrient uptake of crop plants as observed in previous studies (Mumtaz et al. 2018; Arif et al. 2019). The PGPR solubilize inorganic nutrient compounds by releasing low molecular weight organic acids. In this process, bacteria convert the complex compounds into plant available forms that are easily available to plants for uptake (Prabhu et al. 2018; Mumtaz et al. 2019). In present study all strains possess zinc solubilizing potential that might be helpful to reduce the Pb uptake in plants. It has been reported that there exists an antagonistic relationship between Zn and Pb. For example, in a previous study enhanced Zn uptake decreased Pb uptake and accumulation in plants (Musielinska et al. 2016).

Several researchers demonstrated the minimum inhibitory concentration of microorganisms for lead. For example, Saleem et al. (2015) reported Pb tolerant isolates that can use 1800 to 2000 mg L^-1 of Pb. Similarly, Jiang et al. (2017) investigated the tolerance capability of different strains and found that the maximum tolerance concentration for Pb was 1600 mg L^-1. The results of present work showed that Pb adversely affected the growth of bacterial colonies at higher levels. This might be due to toxic effects of Pb on metabolic activities of bacteria (Arif et al. 2019). Metal can cause toxicity by altering enzymes, disturbing structure of proteins and nucleic acid and changing osmotic balance (Pereira et al. 2012). The results regarding lead removal showed that isolates have varying potential for lead removal. Our findings correlated with work of Benmalek and Fardeau (2016) where they reported the Micrococcus spp. as potential metal accumulating agent.

In our study, the selected strains N8, N11, N18, N25, N29 and N35 were identified as B. megaterium N8, B. safensis N11, B. paranthracis N18, B. velezensis N25, B. megaterium N29 and B. subtilis N35, respectively. The identified strains are white and yellow colored with Gram positive nature. These strains are Pb-tolerant; able to grow in lead amended media under both plate and broth assay and have metal removal efficiency. These strains have varied potential to grow under lead stress and showed different PGP traits such as zinc solubilization, phosphate solubilization, siderophore production, exopolysaccharides production, indole acetic acid production, HCN positive and production of hydrolytic enzymes. These also further promoted growth of spinach seedlings in jar trial under lead stress. In previous studies, Jiang et al. (2017) have well documented multiple metal tolerant Bacillus strain jj15 that can grow well at 1600 mg L^-1 of lead. In another study, Abdelkrim et al. (2017) reported Pb tolerant B. simplex and B. megaterium strains which accumulated Pb to their cell surface were able to solubilize phosphorus, produce siderophores and indole acetic acid. Inoculation with these type of lead tolerant bacterial strains significantly improved the biomass of Lathyrus sativus under 0.5 mM Pb reported by Abdelkrim et al. (2018).

Conclusion

Inoculation with Pb-tolerant strains significantly improved growth parameters of spinach seedlings under axenic conditions along with decrease in Pb uptake by spinach plants as compared to control plants. The efficient PGPR strains were identified as B. megaterium (N8), B. safensis (N11), Bacillus spp. (N18), Bacillus spp. (N25), B. megaterium (N29), and B. subtilis (N35) through 16S rRNA sequencing. It is concluded that B. safensis (N11), and Bacillus spp. (N18) are the best strains which not only improved spinach growth but also decreased the Pb uptake by stabilizing it in the root zone. These strains can be a good source of inoculum for growing vegetables by utilizing wastewater as source of irrigation.

Acknowledgement

This work was supported by the Department of Soil Science, University College of Agriculture and Environmental Sciences, the Islamia University of Bahawalpur. The authors also acknowledge the efforts of M. Latif for his services to edit the manuscript.

Author Contributions

All authors equally contributed to this work.

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