Contact us
닫기
Article Search
닫기

Microbiology and Biotechnology Letters

Research Article(보문)

View PDF

Environmental Microbiology  |  Biodegradation and Bioremediation

Microbiol. Biotechnol. Lett. 2023; 51(4): 484-499

https://doi.org/10.48022/mbl.2308.08003

Received: August 10, 2023; Revised: October 10, 2023; Accepted: October 11, 2023

Isolation and Characterization of Plant Growth-Promoting Bacteria for the Phytoremediation of Diesel- and Heavy Metal-Contaminated Soil

Yun-Yeong Lee and Kyung-Suk Cho*

Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea

Correspondence to :
Kyung Suk Cho,      kscho@ewha.ac.kr

Abstract

Go to

Plant growth-promoting (PGP) bacteria can be used as bioresources to enhance phytoremediation through their PGP traits and pollutant removal capacity. In this study, 49 rhizobacteria were primarily isolated from the rhizosphere of tall fescue grown in diesel- and heavy metal-contaminated soil. Their biosurfactant production, phosphate (P) solubilization, and siderophore production were qualitatively and quantitatively evaluated to identify superior PGP bacteria. The optimal conditions for the growth of PGP bacteria and the stability of their PGP traits were a temperature of 35℃, a pH of 7, and 2 days of cultivation time. Four superior PGP bacteria (Pseudomonas sp. NL3, Bacillus sp. NL6, Bacillus sp. LBY14, and Priestia sp. TSY6) were finally selected. Pseudomonas sp. NL3 exhibited superior biosurfactant production and P solubilization. Bacillus sp. NL6 showed the highest P solubilization and superior production of biosurfactants and siderophores. Bacillus sp. LBY14 offered the best siderophore production and impressive P solubilization. Priestia sp. TSY6 had superior capacity for all three PGP traits. Through their secretion of beneficial PGP metabolites, the four bacteria isolated in this study have the potential for use in the phytoremediation of contaminated soil.

Keywords: PGP (plant growth-promoting) bacteria, biosurfactants, P solubility, siderophores, phytoremediation

Graphical Abstract


Introduction

Go to

Rhizobacteria naturally interact with plants in a variety of ways [1]. Some rhizobacteria, which promote plant growth are known as plant growth-promoting (PGP) bacteria, can enhance plant growth through different mechanisms, including nitrogen fixation, phytohormone production, and enzymatic activity [1, 2]. Certain PGP bacteria can reduce pollutant levels or stress-induced damage through biosurfactant production, siderophore production, phosphate (P) solubilization, and the synthesis of stress-relieving enzymes, thereby improving the phytoremediation of plants [1, 2]. Interest in the enhancement of phytoremediation through PGP bacterial inoculation and in the interaction of PGP bacteria with plants have increased over the last few years [3]. Numerous PGP bacteria, such as Pseudomonas, Bacillus, Enterobacter, Klebsiella, Arthrobacter, and Burkholderia, have been widely explored [3].

Biosurfactants are metabolites produced by PGP bacteria, and they consist of diverse compounds, including fatty acids, glycolipids, and lipopeptides [1]. These metabolites reduce the surface tension and viscosity of hydrocarbons while increasing their mobility and bioavailability, thereby improving the efficiency of biodegradation [4]. In addition, biosurfactants form chelates with heavy metals, increasing their solubility and bioavailability in order to desorb them from the soil matrix [1]. Due to these characteristics, biosurfactants are attracting attention as representative metabolites of PGP bacteria with the potential to strengthen the phytoremediation of contaminated soil [1]. Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Lysinibacillus sphaericus can produce the rhamnolipid type of biosurfactants [4, 5], whereas B. subtilis and B. licheniformis can produce surfactin biosurfactants [5].

Phosphate-solubilizing bacteria (PSB) are PGP bacteria that can solubilize insoluble phosphate in soil to supply plants with phosphorus, an essential macronutrient for plant growth [6]. PSBs also enhance phytoremediation efficiency of plants by secreting phytohormones and lowering ethylene levels [7]. Moreover, it has been reported that PSBs can detoxify heavy metals or facilitate the bioavailability of heavy metals for uptake by the plant in metal-contaminated soil [7]. They also secrete heavy metal-chelating low-molecular-weight organic acids, such as lactic, citric, malic, oxalic, acetic, and succinic acids, and thus can remediate heavy metalcontaminated soil [7]. Therefore, P solubility is another important trait of PGP bacteria for the enhancement of phytoremediation. Representative PSBs include bacteria belonging to the genera Pseudomonas, Enterobacter, Bacillus, Rhizobium, Arthrobacter, Burkholderia, and Rahnella [8].

Siderophores are low-molecular-weight iron chelators secreted by plants or microorganisms to improve iron uptake capacity in iron-deficient conditions [2]. Through the production of siderophores, PGP bacteria in the rhizosphere help plants obtain iron, an essential micronutrient [2]. Siderophores have a high affinity not only for Fe2+ but also for divalent cations, such as Cu2+, Pb2+, Cd2+, and Zn2+, and thus can form complexes with heavy metals [9]. The metal-siderophore complexes increase plant growth and the solubility of heavy metals present in soil, enhancing phytoremediation [9]. Siderophores are classified into three groups depending on oxygen ligands: hydroxamates, catecholates, and carboxylates [10]. It has been demonstrated that several Pseudomonas species (including P. fluorescens, P. putida, and P. aeruginosa), Micrococcus luteus, and Streptomyces pilosus can produce the hydroxamate type of siderophore [3], while E. coli, some Rhizobium sp., and some Bacillus species (including B. subtilis, B. cereus, and B. anthracis) can generate catecholates [3, 10]. Some Rhizobium sp. and some Staphylococcus sp. can release carboxylates [10].

In the present study, 49 rhizobacteria were isolated from the rhizosphere of tall fescue cultivated in soil cocontaminated with diesel and heavy metals. The three PGP traits of biosurfactant production, P solubilization, and siderophore production were qualitatively and quantitatively evaluated to screen for superior PGP bacteria. In addition, the optimal experimental conditions in terms of temperature, pH, and cultivation time were investigated to maintain stable PGP traits. The final objective of this study was to secure a bacterial candidate for potential use in enhancing the phytoremediation of diesel- and heavy metal-contaminated soil.

Materials and Methods

Go to

Isolation of rhizobacteria

Rhizobacteria were isolated from the rhizosphere of tall fescue (Festuca arundinacea) grown in soil co-contaminated with diesel and heavy metals for two years. The soil was artificially polluted with diesel, Cu, Pb, and Cd at final concentrations of 30,000 mg-total petroleum hydrocarbons (TPH)·kg-soil-1, 500 mg-Cu·kg-soil-1, 500 mg-Pb·kg-soil-1, and 20 mg-Cd·kg-soil-1, respectively [11]. The pH, water content, and organic matter content of the soil was 6.90 ± 0.06, 40.33 ± 1.40%, and 9.16 ± 1.25%, respectively. To isolate colonies from the tall fescue rhizosphere, five types of agar plates were used: Difco™ M17 Broth, Difco™ Nutrient Broth, Difco™ Luria-Bertani (LB) Broth, Difco™ Plate Count Broth, and Difco™ Tryptic Soy Broth (Becton, Dickinson and Company, USA). An agar of 15 g·l-1 was added to each broth.

For collection, the rhizosphere soil was gently shaken from the tall fescue roots and sieved through a 2-mm mesh. Then, 1 g of sieved soil was mixed with 9 ml of distilled water and serially diluted before 200 μl of each suspension was spread on the agar plates. The plates were cultivated at 30℃ for 24 h. Eight colonies each from the M17, Nutrient, Plate Count, and Tryptic Soy agar plates, and 17 colonies from the LB agar plates. A total of 49 isolates were obtained.

Screening of bacteria with PGP traits

Biosurfactant production was qualitatively evaluated via an emulsification assay [12, 13]. The 49 isolates were pre-cultured using 10 ml of LB broth, and the pre-culture was incubated at 30℃ and 140 rpm for 24 h. For sterilization, diesel oil was dry heated at 160℃ for 24 h. Then, 6 ml of culture medium was added to a sterilized test tube, and an equal volume of sterilized diesel oil was added. The test tube was sealed with a screw cap, vortexed for 2 min to mix, and incubated standing for 24 h at room temperature. After the incubation, the formation of an emulsified layer was observed to screen for biosurfactant-producing strains. To evaluate for P solubilization, a pure colony was spot inoculated on Pikovskaya (PVK) agar containing insoluble Ca3(PO4)2 [14, 15]. The plates were incubated at 30℃ for 7 days, and the formation of a clear halo zone around the colony was monitored to screen strains for phosphate solubilization. To evaluate for siderophore production, a chrome azurol S (CAS) assay was applied [16]. A pure colony was spot inoculated on CAS agar and incubated at 30℃ for 7 days. The formation of an orange halo zone around the colony was observed to screen for siderophoreproducing strains. These evaluations for the three PGP traits of biosurfactant production, P solubilization, and siderophore production led to the screening of 12 strains that each showed two or more capabilities.

Quantitative evaluation to establish optimal conditions for PGP traits

Optimal condition tests for the PGP traits of biosurfactant production, P solubilization, and siderophore production were conducted using the twelve selected strains. These twelve colonies were pre-cultured in 30 ml of LB broth and incubated at 30℃ and 140 rpm for 24 h. Then, 0.3 ml of cell suspension was inoculated into 30 ml of LB broth (1%, v/v) and cultivated at 30℃ and 140 rpm for 24 h. The cell suspension was centrifuged at 4,000 rpm for 5 min and then washed with sterile water twice. The cell pellet was resuspended in LB broth at the final OD600 of 0.6. The cell culture in which OD600 was adjusted to 0.6 was used to evaluate PGP traits depending on different conditions. First, the PGP traits were quantitatively evaluated at different cultivation temperatures (25, 30, 35, and 40℃) for 24 h. Next, PGP traits were investigated at different pH levels of the culture medium. The pH of the culture medium was adjusted to 5, 6, 7, and 8 before incubation at 35℃ for 24 h. Finally, PGP traits for different cultivation times (1−6 days) were explored at a temperature of 35℃ and a pH of 7.

The biosurfactant production of the 12 strains was quantitatively calculated using the emulsification assay and emulsification index (E24) [12, 13]. For this analysis, 6 ml of cell culture (OD600 of 0.6) was mixed with an equal volume of sterilized diesel oil and incubated at a specific temperature (25−40℃) for 1−6 days. E24 was calculated using Eq. (1) [12, 13]:

E24(%)=He/Hs×100

where He is the height of the emulsified layer (cm) and Hs is the total height of the solution (cm).

The P solubilization of the twelve strains was quantitatively investigated in the broth medium using the colorimetric method [6, 17]. The broth medium contained 1 g·l-1 of glucose, 2 g·l-1 of NaCl, 0.5 g·l-1 of MgCl2· 6H2O, 0.025 g·l-1 of MgSO4·7H2O, 0.02 g·l-1 of KCl, 0.01 g·l-1 of (NH4)2SO4, and 0.5 g·l-1 of insoluble Ca3(PO4)2 [6]. For the analysis, 0.3 ml of cell culture (OD600 of 0.6) was inoculated into 30 ml of broth medium (1%, v/v) and then incubated at a specific temperature (25−40℃) for 1−6 days. After the incubation, the mixture was centrifuged at 4,000 rpm for 15 min, and the supernatant was obtained. The soluble phosphate concentration of the supernatant was measured using the ascorbic acid method in accordance with the Korean Standard Water Analysis Method (ES.04360.2c) [18]. The absorbance was measured using a UV/Vis spectrometer (Biochrom Ltd., UK) at 880 nm, and the quantity of soluble phosphate was expressed as μg-PO4-P·mg-dry cell weight (DCW)-1.

The siderophore production of the twelve strains was quantitatively measured by colorimetric analysis [19, 20]. Briefly, 0.3 ml of cell culture (OD600 of 0.6) was cultured in 30 ml of nutrient broth (1%, v/v) at a specific temperature (25−40℃) for 1−6 days. Our previous study described the procedure in more detail [11]. The absorbance was measured using a UV/Vis spectrometer (Biochrom Ltd.) at 700 nm, and the quantity of siderophores produced was expressed as μM-benzoic acid (BA)·mg-DCW-1.

Identification of four PGP bacteria

From the twelve strains, four strains (NL3, NL6, LBY14, and TSY6) with superior PGP traits were finally selected. They were identified by a 16S rRNA sequencing analysis. To obtain genomic DNA, a colony was vortexed with 30 μl of sterilized water for 30 s. The suspension was heat-treated in a heating block at 95℃ for 15 min and then centrifuged at 11,000 rpm for 30 s [11]. The procedure was repeated three times. For the 16S rRNA sequencing, a bacterial universal primer set consisting of 27F (5’-AGA GTT TGA TCM TGG CTC AG-3’) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3’) was utilized [21]. The polymerase chain reaction (PCR) mixture contained 5 μl of 10× PCR buffer (Genenmed Inc., Korea), 4 μl of 2.5 mM dNTPs (Genenmed Inc.), 0.4 μl of bovine serum albumin (BSA), 1 μl of 10 μM each of forward/reverse primer, 1 U of ACE Taq polymerase (Genenmed Inc.), and 2 μl of genomic DNA template in a total reaction volume of 50 μl. PCR was performed using a T100™ Thermal Cycler (Bio-Rad Laboratories, USA). Initial denaturation was performed at 94℃ for 3 min and followed by 30 cycles of denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, and final extension at 72℃ for 5 min [22]. The sequences of the PCR products were analyzed by Macrogen Inc. (Korea) and compared with entries from the National Center for Biotechnology Information (NCBI) GenBank database Nucleotide BLAST. The obtained sequences were deposited into the NCBI Gen-Bank database under accession number OQ625802 (strain NL3), OQ625779 (strain NL6), OQ625809 (strain LBY14), and OQ625781 (strain TSY6).

Results

Go to

Qualitative evaluation of 49 isolates for PGP traits

A total of 49 isolates (ML1-ML8, NL1-NL8, LBY1-LBY17, PCY1-PCY8, and TSY1-8) were obtained from the tall fescue rhizosphere. These isolates were qualitatively evaluated for the PGP traits of biosurfactant production, P solubilization, and siderophore production, as shown in Tables 1 and S1. To evaluate biosurfactant production, the height of the emulsified layer between the cell culture and diesel layers was measured. An emulsified layer height of less than 1 cm was evaluated as weak (+), 1−2 cm as moderate (++), and more than 2 cm as strong (+++, Table 1). A total of 22 isolates showed positive biosurfactant production, of which 8 isolates (ML3, ML5, ML7, NL3, LBY13, LBY16, PCY6, and PCY7) were relatively superior (Table 1).

Table 1 . Qualitative evaluation of 49 isolates for PGP traits.

Medium used for isolationIsolate #Biosurfactant productionaP solubilizationbSiderophore productionb
M17 agarML1+--
ML2---
ML3+++++++
ML4---
ML5+++--
ML6---
ML7+++++++
ML8--+
Nutrient agarNL1-+++
NL2---
NL3+++++
NL4--+
NL5+-+
NL6++++++
NL7++--
NL8+--
LB agarLBY1---
LBY2---
LBY3---
LBY4--+++
LBY5--+
LBY6---
LBY7-++-
LBY8---
LBY9+--
LBY10+--
LBY11---
LBY12+--
LBY13+++++
LBY14++-++
LBY15++--
LBY16+++++
LBY17---
Plate Count agarPCY1--+
PCY2+--
PCY3--+
PCY4---
PCY5---
PCY6+++-+
PCY7+++--
PCY8-++-
Tryptic Soy agarTSY1---
TSY2+--
TSY3+-+
TSY4+--
TSY5---
TSY6-++++
TSY7---
TSY8---

a The height of the emulsified layer was measured. +, weak (< 1 cm); ++, moderate (1-2 cm); +++, strong (≥ 2 cm).

b The length between the colony edge and halo zone was measured. +, weak (< 0.1-0.3 cm); ++, moderate (0.3-0.5 cm); +++, strong (≥ 0.5 cm). Gray shading indicates the primarily selected twelve strains with positive results for two or more traits.

For P solubilization, a clear halo zone was observed around a colony. A length between the colony edge and halo zone of 0.1−0.3 cm was evaluated as weak (+), 0.3− 0.5 cm as moderate (++), and more than 0.5 cm as strong (+++, Table 1). A total of 10 isolates (ML3, ML7, NL1, NL3, NL6, LBY7, LBY13, LBY16, PCY8, and TSY6) showed positive P solubilization, with ML7 demonstrating the greatest P solubilization (Table 1).

An orange halo zone around a colony was observed to evaluate siderophore production. A length between the colony edge and halo zone of 0.1−0.3 cm was evaluated as weak (+), 0.3−0.5 cm as moderate (++), and more than 0.5 cm as strong (+++, Table 1). A total of 18 isolates (ML3, ML7−8, NL1, NL3−6, LBY4−5, LBY13−14, LBY16, PCY1, PCY3, PCY6, TSA3, and TSY6) exhibited positive siderophore production, of which NL6, LBY4, and TSY6 showed the highest siderophore production (Table 1).

Of the 49 original isolates, twelve isolates with positive findings for two or more traits were first selected. Six isolates (ML3, ML7, NL3, NL6, LBY13, and LBT16) showed positive results for all three traits, and the other six isolates (NL1, NL5, LBY14, PCY6, TSY3, and TSY6) showed positive results for two traits (Tables 1 and S1).

Identifying optimal conditions for PGP traits through quantitative evaluation

Fig. 1 shows the quantitative results for the PGP traits of the twelve screened strains at different cultivation temperature (25−40℃). Five strains (NL1, NL3, NL5, NL6, and TSY6) showed a relatively high E24 index, which represents high biosurfactant production (Fig. 1A). Most of the strains exhibited the highest E24 values at 35℃. While no considerable differences were observed in the soluble P concentrations among strains (Fig. 1B), there were differences in soluble P concentrations depending on the cultivation temperature. In general, the highest soluble P concentrations were measured at 35℃, indicating that the highest P solubilization was achieved at 35℃. Finally, siderophore concentration is shown in Fig. 1C. It was difficult to find a distinct trend in siderophore concentration, as the optimal temperature differed by strain. Three strains (ML7, NL5, and LBY13) produced the highest sidero-phore concentrations at 30℃, compared with 35℃ for the other three strains (NL6, LBY14, and LBY16). In a comprehensive analysis of the E24, soluble P concentration, and siderophore concentration, the optimal temperature for PGP traits was thought to be 35℃.

Figure 1.Comparison of PGP traits at different cultivation temperatures. (A) E24 (%), (B) soluble P concentration, and (C) siderophore concentration.

Fig. 2 shows the quantitative PGP traits at different levels of pH (5−8) in the culture medium. The cultivation temperature was fixed at 35℃, according to the results in Fig. 1. Relatively high E24 values were observed at pH levels of 7−8, while most strains did not produce biosurfactants under the weakly acidic conditions of a pH of 5 (Fig. 2A). In the pH range of 7−8, strains NL1, NL3, NL5, NL6, and TSY6 exhibited relatively high E24 values. The soluble P concentration was relatively high at a pH of 6−7, while it was lowest at a pH of 5 (Fig. 2B). Except at a pH of 5, there were no considerable differences in soluble P concentration depending on pH and strain. Half the strains did not exhibit siderophore production at a pH of 5 (Fig. 2C), but most of the strains produced the highest siderophore concentrations at a pH of 7. In general, the optimal pH was different for each PGP trait. There were no considerable differences in E24 between pH levels of 7 and 8 or in soluble P concentrations between pH levels of 6 and 7. Siderophore concentration was highest at a pH of 7. Given the common range, it was considered most appropriate to set the pH in the culture medium to 7 for the phytoremediation activities.

Figure 2.Comparison of PGP traits at different pH levels of the culture medium. (A) E24 (%), (B) soluble P concentration, and (C) siderophore concentration.

Fig. 3 demonstrates the E24 index at different cultivation times. All strains were cultivated for 6 days. The cultivation temperature and pH level were 35℃ and 7, respectively, as shown in Figs. 1 and 2. Strain ML7 did not produce biosurfactants, while four strains (NL1, NL3, NL6, and TSY6) showed relatively high E24 values. Multiple comparisons of these four strains were performed, and there were no significant differences in their E24 values over the cultivation times (p > 0.05). Fig. 4 displays the changes in soluble P concentration and DCW at different cultivation times. DCW was highest on day 2 and then continuously decreased. The soluble P concentration generally increased as the cultivation time elapsed. However, it was thought that the soluble P concentration per unit of DCW was overestimated due to the DCW decreasing after 2 days. Of the twelve strains, four strains (NL3, NL6, LBY14, and LBY16) had relatively high soluble P concentrations on day 2. Fig. 5 indicates the changes in siderophore concentration and DCW at different cultivation times. DCW showed the highest values on days 2−3 of cultivation and then gradually decreased. The siderophore concentration continuously increased over time, with the highest concentration observed on days 5−6. However, like the P solubilization, the siderophore concentration per unit of DCW was thought to be overestimated because the DCW decreased after days 2−3. According to a comprehensive look at the changes in DCW and PGP traits, two days of cultivation time was considered most suitable for the growth and activity of the strains.

Figure 3.Comparison of E24 (%) index at different cultivation times.

Figure 4.Comparison of dry cell weight and soluble P concentrations at different cultivation times. Strain (A) ML3, (B) ML7, (C) NL1, (D) NL3, (E) NL5, (F) NL6, (G) LBY13, (H) LBY14, (I) LBY16, (J) PCY6, (K) TSY3, and (L) TSY6.

Figure 5.Comparison of dry cell weight and siderophore concentration at different cultivation times. Strain (A) ML3, (B) ML7, (C) NL1, (D) NL3, (E) NL5, (F) NL6, (G) LBY13, (H) LBY14, (I) LBY16, (J) PCY6, (K) TSY3 and (L) TSY6.

Final selection and identification of four PGP bacteria

The average values of PGP traits for the twelve bacteria were calculated in Table 2 using the results from the optimal temperature: 35℃ (Fig. 1), a pH of 7 (Fig. 2), and day 2 of cultivation time (Figs. 3−5). The average E24, soluble P concentration, and siderophore concentration are shown in Table 2, and the raw data are in Tables S2−S4. Strains NL1, NL3, NL5, NL6, and TSY6 showed consistently high E24 (high biosurfactant production), with an average of 23.8−32.4% (Tables 2 and S2). Although it was difficult to select strains that consistently maintained a high P solubilization (Table S3), P solubilization was highest with strain NL6 (average of 148.7 μg-PO4-P·mg-DCW-1) relative to the twelve strains (Table 2). It was also difficult to screen for strains that steadily exhibited superior siderophore production (Table S4). However, strains LBY14 (44.0 μg-BA·mg-DCW-1), PCY6 (44.8 μg-BA·mg-DCW-1), and TSY3 (44.6 μg-BA·mg-DCW-1) produced a relatively high siderophore concentration on average (Table 2).

Table 2 . The average values of PGP traits (E24, soluble P concentration, and siderophore concentration) of the 12 selected bacteria.

Isolate #Average E24 (%)Average soluble P concentration (μg-PO4-P·mg-DCW-1)Average siderophore concentration (μg-BA·mg-DCW-1)
ML311.3 ± 3.2117.5 ± 83.816.7 ± 14.1
ML71.7 ± 1.4121.8 ± 25.422.6 ± 20.4
NL132.4 ± 7.284.4 ± 40.19.1 ± 5.1
NL331.1 ± 7.3119.6 ± 52.921.5 ± 19.3
NL523.8 ± 11.193.4 ± 52.823.1 ± 22.3
NL630.1 ± 10.1148.7 ± 44.131.3 ± 10.4
LBY138.2 ± 1.6108.2 ± 19.238.9 ± 11.8
LBY148.8 ± 2.5125.9 ± 43.644.0 ± 18.4
LBY167.8 ± 4.7129.1 ± 37.736.8 ± 11.5
PCY66.5 ± 3.865.3 ± 23.644.8 ± 23.8
TSY310.9 ± 2.285.6 ± 47.344.6 ± 35.3
TSY624.9 ± 8.690.6 ± 46.932.8 ± 24.0

Gray shading indicates the final four bacteria with superior PGP traits.

Four bacterial strains (NL3, NL6, LBY14, and TSY6) with superior PGP traits were finally selected. Although strain NL3 showed a relatively weak siderophore production (21.5 μg-BA·mg-DCW-1), its biosurfactant production (31.1%) and P solubilization (119.6 μg-PO4- P·mg-DCW-1) were considerable. Of the twelve strains, NL6 exhibited the best P solubilization (148.7 μg-PO4- P·mg-DCW-1) and had a superior biosurfactant (30.1%) and siderophore production (31.3 μg-BA·mg-DCW-1). Although the biosurfactant production of LBY14 was relatively low (8.8%), the strain showed a strong P solubilization (125.9 μg-PO4-P·mg-DCW-1) and the highest siderophore production (44.0 μg-BA·mg-DCW-1). Finally, strain TSY6 performed well for all traits (24.9% for biosurfactant production, 90.6 μg-PO4-P·mg-DCW-1 for P solubilization, and 32.8 μg-BA·mg-DCW-1 for siderophore production). The final four bacterial strains were identified as Pseudomonas sp. NL3, Bacillus sp. NL6, Bacillus sp. LBY14, and Priestia sp. TSY6 (Fig. 6).

Figure 6.The phylogenetic tree of the final selected strains (NL3, NL6, LBY14, and TSY6) based on 16S rRNA sequences. The representative sequences were obtained from the NCBI GenBank Database. Bootstrap values are shown at the branch points. The scale bar indicates 0.01 substitutions per site.

Discussion

Go to

In this study, bacteria from the Pseudomonas, Bacillus, and Priestia genera were isolated from the tall fescue rhizosphere in diesel- and heavy metal-contaminated soil, and their PGP traits (biosurfactant production, P solubilization, and siderophore production) were explored. Biosurfactants not only degrade hydrocarbons directly, but also increase the bioavailability of heavy metals. P solubilization helps plant growth by supplying insoluble phosphate in the form of soluble phosphorus, while reducing the toxicity of heavy metals and increasing their bioavailability. Finally, siderophores can improve the bioavailability of heavy metals, thereby enhancing phytoremediation. The three PGP traits employed in this study directly affect the pollutants level in soil, especially by lowering the toxicity and increasing bioavailability of heavy metals to facilitate uptake by plants. Through these mechanisms, the plant biomass also increases, and consequently, it helps to strengthen the phytoremediation by plants in contaminated soil.

Of the four strains, Pseudomonas sp. NL3 exhibited superior biosurfactant production and P solubilization, but the lowest siderophore production. Pseudomonas is one of the most representative genera for PGP bacteria, with its members commonly found in rhizospheric soil [3]. Several Pseudomonas species, such as P. aeruginosa, P. fluorescens, and P. putida, have been reported to exhibit strong PGP traits [23]. Many studies have focused on the biosurfactant production of P. aeruginosa in particular because of its high production yield, short incubation time, and easy cultivation [24]. Accordingly, numerous studies have used P. aeruginosa to investigate the optimization of biosurfactant production across a wide range of temperatures (25−40℃), pH levels (5−10), and cultivation times (40 h−10 d) [24]. For optimal bacterial growth and biosurfactant production by P. aeruginosa, the pH of the culture medium should be adjusted to a neutral range (7−7.5) [24]. Although P. aeruginosa is able to survive at a wide temperature range of 4−42℃, the optimal temperature has been reported to be 37℃ [24]. Previous reports have also examined the optimization of biosurfactant production by other Pseudomonas species [25−27]. For example, in a study of P. cepacia, P. acidovorans, P. picketii, and P. fluorescens, Rocha e Silva et al. found that P. cepacia achieved maximum biosurfactant production at 30℃ after 14 h [25]. In other studies, P. nitroreducens, isolated from petroleum-contaminated soil, exhibited high biosurfactant production at 30℃ [26], whereas P. fluorescens from freshwater showed maximum biosurfactant production at 28℃ after 92 h [27].

P solubilization is another important PGP trait shown by Pseudomonas species. P. fluorescens, P. putida, and P. chlororaphis are known to have superior P solubilization [23, 28]. Several previous studies have explored the optimization of P solubilization by Pseudomonas strains. Oteino et al. cultivated several Pseudomonas strains and found P. fluorescens L225 and Pseudomonas sp. L132 to show the highest P solubilization at 30℃ [29]. In addition, these strains promoted plant growth by secreting gluconic acid when they were inoculated into pea plants (Pisum sativum L) [29]. Jha et al. cultivated eighty Pseudomonas strains at 28℃ for 3 days to screen for P solubilization, reporting that the P. plecoglossicida FP12 and P. mosselii FP13 strains exhibited relatively superior P solubilization [30].

The genus Pseudomonas not only is abundant in the rhizosphere of plants but also can produce a large amount of metal-chelating siderophores under ironlimiting conditions [31]. Several Pseudomonas species, such as P. aeruginosa, P. fluorescens, and P. putida, have been reported to produce hydroxamate siderophores [3]. Numerous previous studies have investigated the enhancement of heavy metal uptake and plant phytoremediation by Pseudomonas inoculation [32]. Sah et al. evaluated siderophore production using fourteen Pseudomonas strains, of which P. aeruginosa RSP5 exhibited the highest production and transported more iron to the maize plant, thereby increasing plant growth and iron content in plant tissues [33]. In another study, a P. aeruginosa strain capable of producing siderophores was inoculated into Solanum nigrum seedlings, significantly increasing plant growth and Cd translocation [34].

In the present study, two Bacillus strains (NL6 and LBY14) exhibited strong PGP traits. Bacillus sp. NL6 had the highest P solubilization and superior production of biosurfactants and siderophores. On the other hand, Bacillus sp. LBY14 showed the lowest biosurfactant production but considerable P solubilization and the highest siderophore production. Along with the genus Pseudomonas, members of the genus Bacillus are some of the most abundant PGP bacteria in the rhizosphere. Thus, research on the PGP abilities of Bacillus species has been widely conducted over the years [3]. Bacillus species generally produce spores, making them viable at extreme temperature, pH, and osmotic conditions and highly resistant to unfavorable environments [35]. This environmental adaptability makes the Bacillus species an attractive alternative for field application [35]. Bacillus species produce the surfactin type of biosurfactants, and several Bacillus species, including B. subtilis, B. cereus, and B. licheniformis, are representative biosurfactant producers [36]. Many previous studies have evaluated the biosurfactant production of Bacillus over a wide range of environmental conditions because of their environmental resistance. B. subtilis TPCC 1696 [37], B. methylotrophicus USTBa [38], and B. licheniformis R2 [39] exhibited stable biosurfactant production and hydrocarbon degradability across a wide range of pH levels (2−12) and temperatures (15−100℃). Although Bacillus can secrete biosurfactants across a wide range of environmental conditions, previous studies have also demonstrated Bacillus to display the highest biosurfactant production under similar environmental conditions to those in this study. Priya and Usharani isolated B. subtilis from oil-contaminated soil, and the optimal temperature for biosurfactant production was 35−40℃ [40]. In other studies, B. licheniformis R2 effectively produced biosurfactants at 30℃ and a pH of 6.5−7.2 [41], whereas Bacillus brevis had optimal conditions for biosurfactant production at 33℃, a pH of 8, and 10 days of incubation [42].

Several Bacillus species, including B. cereus, B. subtilis, B. circulans, and B. pumilus, have been reported to exhibit P solubilization [43], with quite different optimal conditions reported depending on strain. Mehta et al. isolated B. subtilis CB8A from the apple rhizosphere and explored its PGP traits for 72 h at a range of 30−45℃ in temperature and 7−9 in pH level [44]. Maximum P solubilization was achieved at 37℃ and a pH of 7 [44], which was consistent with the results of this study. In addition, B. subtilis CB8A produced gluconic and citric acids as the major organic acids for P solubilization [44]. Rahman et al. reported that the optimal conditions for P solubilization by Bacillus sp. isolated from saline soil were a temperature of 30℃, pH of 5, and 6 days of cultivation [45]. Banerjee et al. isolated Bacillus sp. TRSB16 from the tomato rhizosphere and explored its P solubilization under stressful conditions of pH (8−11) and temperature (37−50℃) [46]. The concentration of phosphorus solubilized by strain TRSB16 was highest at a pH of 10 and temperature of 37℃ [46].

B. anthracis, B. cereus, B. licheniformis, and B. subtilis are well-known Bacillus species that secrete siderophores, mainly catecholates [43, 47]. As there were for biosurfactants and P solubilization, there are numerous studies on siderophore production by Bacillus species and the enhancement of phytoremediation. The siderophore production of B. subtilis CB8A isolated from the apple rhizosphere was explored at a temperature of 30− 45℃ and pH of 7−9 [44]. Maximum siderophore production (50%) was observed at a temperature of 37℃ and a pH of 7 [44], which was consistent with the results of this study. Bacillus sp. PZ-1 showed maximum siderophore production (91%) at a pH of 6.18, and Pb uptake from soil was improved when strain PZ-1 was inoculated into Brassica juncea [48]. In another study, B. subtilis CAS15 significantly improved the height of pepper plants by producing siderophores at a temperature of 28℃ [49].

Priestia sp. TSY6 showed generally good results for the three PGP traits in the present study. Although the genus Priestia is not studied as often as Pseudomonas and Bacillus, several studies evaluated its PGP traits, with most focusing on the PGP traits of Priestia megaterium [50]. P. megaterium, which was formerly known as Bacillus megaterium [50], is able to secrete phytohormone and has been reported to enhance the growth of various plants, such as tomato, maize, mustard, rice, and soybean [50]. Apart from P. megaterium, the PGP traits and plant growth promotion of other Priestia species, such as P. koreensis [51], P. endophytica [52], and P. aryabhattai [53], were reported by a small number of studies. Recently, a few studies have explored biosurfactant production by members of the genus Priestia, with most of them investigating P. megaterium. Sandhu et al. isolated P. megaterium sp. MAPB-27 from soil near a steel plant. Similar to the results in this study, the optimal conditions for growth were 30℃ and a pH of 7 [54]. Strain MAPB-27 also showed an emulsification capacity of 44% and degradation of 53% for chlorinated hydrocarbons [54]. After its isolation from sediment, P. megaterium sp. ZS16 generated lipopeptide biosurfactants at 37℃ and showed emulsification activity with petroleum oil [55]. In another study, two Priestia sp. strains were isolated from crude oil-contaminated soil, and various hydrocarbons (benzene, toluene, xylene, kerosene, and diesel oil) were emulsified via biosurfactant production [56].

Several studies have examined the P solubilization of members of the genus Priestia. For example, P. megaterium is a well-known PSB that helps plants to utilize phosphorus from the soil by secreting organic acids and extracellular enzymes [57]. Hu et al. isolated P. megaterium A6 from the rhizosphere of oilseed rape and demonstrated that this strain was able to solubilize the mineral phosphate by secreting organic acids (succinic and citric acids) [58]. Jiang et al. also reported that P. megaterium YM13 exhibited P solubilization and improved the germination and growth of peanut seeds [59]. Regarding other Priestia species, P. aryabhattai BPR-9 isolated from wheat plant tissues [53] and P. endophytica SK1 from the fenugreek rhizosphere [52] have been reported to solubilize phosphate. In addition, both strains significantly promoted plant germination and biomass compared with a non-inoculated control [52, 53].

As described above, siderophores form complexes with heavy metals, thereby assisting phytoremediation in heavy metal-contaminated soil [9]. Several Priestia strains have been reported to use both siderophore production and heavy metal resistance to enhance phytoremediation. P. aryabjattai BRP-9 isolated from the tissues of wheat plants exhibited siderophore production of 53% at 28℃ and superior resistance to five heavy metals (Cd, Cr, Cu, Pb, and Hg) [53]. Priestia sp. LWS1 isolated from selenium mine soil also exhibited siderophore production (0.23 mg· l-1) and Se-tolerance (270 mg·l-1 of EC50) at 30℃ [60]. The siderophore production of P. megaterium P12 [61] and P. kreensis LV19 [51] have been explored as well.

In the present study, four superior PGP bacteria were identified from the rhizosphere of tall fescue grown in diesel- and heavy metal-contaminated soil. In particular, it was demonstrated that genus Priestia, which has not been often studied before, showed superior overall PGP capabilities. Furthermore, the optimum conditions for their growth and the stability of PGP traits were simultaneously investigated. On the other hand, this study has a limitation in that it was performed in a controlled pure culture and the application experiments using the strains are insufficient. Phytoremediation takes place under an uncontrolled natural environment, and it occurs through complex interactions of the microbial communities in rhizosphere. Therefore, a further study is needed on how the identified PGP bacteria affect phytoremediation by inoculation into plants growing in the actual contaminated soil. Nevertheless, the results obtained in this study can be used as a basic knowledge to strengthen phytoremediation in co-contaminated soil with diesel and heavy metals.

Conclusion

Go to

From the rhizosphere of tall fescue cultivated in soil co-contaminated with diesel and heavy metals, 49 rhizobacteria were isolated. Afterward, twelve PGP bacterial strains with positive findings for the three PGP traits of biosurfactant production, P solubilization, and siderophore production were screened. According to a quantitative evaluation of PGP traits using the twelve strains, the optimal conditions for PGP activities were a temperature of 35℃, a pH of 7, and 2 days of cultivation time. Of the twelve PGP bacterial strains, four strains (Pseudomonas sp. NL3, Bacillus sp. NL6, Bacillus sp. LBY14, and Priestia sp. TSY6) with superior PGP abilities were finally selected. Pseudomonas sp. NL3 had a relatively weak siderophore production (21.5 μg- BA·mg-DCW-1) but great biosurfactant production (31.1%) and P solubilization (119.6 μg-PO4-P·mg-DCW-1). Bacillus sp. NL6 showed the best P solubilization (148.7 μg-PO4-P·mg-DCW-1) and excellent biosurfactant (30.1%) and siderophore (31.3 μg-BA·mg-DCW-1) production. Although Bacillus sp. LBY14 exhibited the lowest biosurfactant production (8.8%), it showed considerable P solubilization (125.9 μg-PO4-P·mg-DCW-1) and the highest siderophore production (44.0 μg-BA·mg-DCW-1). Finally, Priestia sp. TSY6 exhibited superior overall PGP capabilities, with a biosurfactant production of 24.9%, P solubilization of 90.6 μg-PO4-P·mg-DCW-1, and siderophore production of 32.8 μg-BA·mg-DCW-1. The four PGP bacterial strains identified in this study can be utilized as biostimulators to strengthen phytoremediation by inoculation into plants growing in contaminated soil.

Highlights

Go to

Four PGP bacteria were isolated from the tall fescue rhizosphere of contaminated soil.

Stable PGP traits were achieved at 35℃, a pH of 7, and 2 days of incubation.

Pseudomonas sp. NL3 had considerable biosurfactant production and P solubilization.

Bacillus sp. LBY14 showed superior P solubilization and siderophore production.

Bacillus sp. NL6 and Priestia sp. TSY6 performed well for all PGP traits.

Acknowledgments

Go to

This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government, the Ministry of Science and ICT (MICT) (2019R1A2C2006701 & 2022R1A2C2006615).

References

Go to
  1. Ullah A, Heng S, Munis MFH, Fahad S, Yang X. 2015. Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: a review. Environ. Exp. Bot. 117: 28-40.
    CrossRef
  2. Saeed Q, Xiukang W, Haider FU, Kučerik J, Mumtaz MZ, Holatko J, et al. 2021. Rhizosphere bacteria in plant growth promotion, biocontrol, and bioremediation of contaminated sites: A comprehensive review of effects and mechanisms. Int. J. Mol. Sci. 22: 10529.
    Pubmed KoreaMed CrossRef
  3. Saharan BS, Nehra V. 2011. Plant growth promoting rhizobacteria: a critical review. Life Sci. Med. Res. 21: 1-30.
  4. Karlapudi AP, Venkateswarulu TC, Tammineedi J, Kanumuri L, Ravuru BK, Dirisala V, et al. 2018. Role of biosurfactants in bioremediation of oil pollution-a review. Petroleum 4: 241-249.
    CrossRef
  5. Mulligan CN. 2021. Sustainable remediation of contaminated soil using biosurfactants. Front. Bioeng. Biotechnol. 9: 635196.
    Pubmed KoreaMed CrossRef
  6. Teymouri M, Akhtari J, Karkhane M, Marzban A. 2016. Assessment of phosphate solubilization activity of rhizobacteria in mangrove forest. Biocatal. Agric. Biotechnol. 5: 168-172.
    CrossRef
  7. Ahemad M. 2015. Phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: a review. 3 Biotech 5: 111-121.
    Pubmed KoreaMed CrossRef
  8. Rawat P, Das S, Shankhdhar D, Shankhdhar SC. 2021. Phosphatesolubilizing microorganisms: mechanism and their role in phosphate solubilization and uptake. J. Soil Sci. Plant Nutr. 21: 49-68.
    CrossRef
  9. Ahmed E, Holmström SJM. 2014. Siderophores in environmental research: roles and applications. Microb. Biotechnol. 7: 196-208.
    Pubmed KoreaMed CrossRef
  10. Saha M, Sarkar S, Sarkar B, Sharma BK, Bhattacharjee S, Tribedi P. 2016. Microbial siderophores and their potential applications: a review. Environ. Sci. Pollut. Res. 23: 3984-3999.
    Pubmed CrossRef
  11. Lee SY, Lee YY, Cho KS. 2023. Effect of Novosphingobium sp. CuT1 inoculation on the rhizoremediation of heavy metal- and dieselcontaminated soil planted with tall fescue. Environ. Sci. Pollut. Res. 30: 16612-16625.
    Pubmed CrossRef
  12. Barakat KM, Hassan SWM, Darwesh OM. 2017. Biosurfactant production by haloalkaliphilic Bacillus strains isolated from Red Sea, Egypt. Egypt. J. Aquat. Res. 43: 205-211.
    CrossRef
  13. Morales-Guzmán G, Ferrera-Cerrato R, Rivera-Cruz MC, Torres-Bustillos LG, Arteaga-Garibay RI, Mendoza-López MR, et al. 2017. Diesel degradation by emulsifying bacteria isolated from soils polluted with weathered petroleum hydrocarbons. Appl. Soil Ecol. 121: 127-134.
    CrossRef
  14. Mihalache G, Mihasan M, Zamfirache MM, Stefan M, Raus L. 2018. Phosphate solubilizing bacteria from runner bean rhizosphere and their mechanism of action. Rom. Biotechnol. Lett. 23: 13853-13861.
  15. Verma PP, Kaur M. 2015. Characterization of the mineral phosphate solubilizing activity of fluorescent Pseudomonas sp. isolated from rhizosphere of apple. Trends Biosci. 8: 6600-6604.
  16. Louden BC, Haarmann D, Lynne AM. 2011. Use of blue agar CAS assay for siderophore detection. J. Microbiol. Biol. Educ. 12: 51-53.
    Pubmed KoreaMed CrossRef
  17. Elhaissoufi W, Khourchi S, Ibnyasser A, Ghoulam C, Rchiad Z, Zeroual Y, et al. 2020. Phosphate solubilizing rhizobacteria could have a stronger influence on wheat root traits and aboveground physiology than rhizosphere P solubilization. Front. Plant Sci. 11: 979.
    Pubmed KoreaMed CrossRef
  18. Korean Ministry of Environment. 2017. Korean Standard Water Analysis Method, Revised in 2022. Available from https://www.law.go.kr/DRF/lawService.do?OC=me_pr&target=admrul&ID=2100000209158&type=HTML&mobileYn=. Accessed Aug. 7, 2023.
  19. Hwang JS, Song HG. 2020. Antifungal activity of Bacillus subtilis isolates against toxigenic fungi. Korean J. Microbiol. 56: 28-35.
  20. Nagarajkumar M, Bhaskaran R, Velazhahan R. 2004. Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen. Microbiol. Res. 159: 73-81.
    Pubmed CrossRef
  21. Ashfaq M, Afzal A, Javed MA, Ali M, Rasool B, Shaheen S, et al. 2021. Identification and pathogenic characterization of bacteria causing rice grain discoloration in Pakistan. Int. J. Biol. Biotechnol. 18: 239-246.
  22. Kim TG, Moon KE, Yun J, Cho KS. 2013. Comparison of RNA- and DNA-based bacterial communities in a lab-scale methanedegrading biocover. Appl. Microbiol. Biotechnol. 97: 3171-3181.
    Pubmed CrossRef
  23. Singh P, Singh RK, Zhou Y, Wang J, Jiang Y, Shen N, et al. 2022. Unlocking the strength of plant growth promoting Pseudomonas in improving crop productivity in normal and challenging environments: a review. J. Plant Interact. 17: 220-238.
    CrossRef
  24. Eslami P, Hajfarajollah H, Bazsefidpar S. 2020. Recent advancements in the production of rhamnolipid biosurfactants by Pseudomonas aeruginosa. RSC Adv. 10: 34014-34032.
    Pubmed KoreaMed CrossRef
  25. Rocha e Silva NMP, Rufino RD, Luna JM, Santos VA, Sarubbo LA. 2014. Screening of Pseudomonas species for biosurfactant production using low-cost substrates. Biocatal. Agric. Biotechnol. 3: 132-139.
    CrossRef
  26. Onwosi CO, Odibo FJC. 2012. Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from soil. World J. Microbiol. Biotechnol. 28: 937-942.
    Pubmed CrossRef
  27. Janek T, Łukaszewicz M, Rezanka T, Krasowska A. 2010. Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard. Bioresour. Technol. 101: 6118-6123.
    Pubmed CrossRef
  28. Nadeem SM, Naveed M, Ayyub M, Khan MY, Ahmad M, Zahir ZA. 2016. Potential, limitations and future prospects of Pseudomonas spp. for sustainable agriculture and environment: A review. Soil Environ. 35: 106-145.
  29. Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, et al. 2015. Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front. Microbiol. 6: 745.
    Pubmed KoreaMed CrossRef
  30. Jha BK, Pragash MG, Cletus J, Raman G, Sakthivel N. 2008. Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii. World J. Microbiol. Biotechnol. 25: 573-581.
    CrossRef
  31. Dorjey S, Dolkar D, Sharma R. 2017. Plant growth promoting rhizobacteria Pseudomonas: a review. Int. J. Curr. Microbiol. Appl. Sci. 6: 1335-1344.
    CrossRef
  32. David SR, Geoffroy VA. 2020. A review of asbestos bioweathering by siderophore-producing Pseudomonas: a potential strategy of bioremediation. Microorganisms 8: 1870.
    Pubmed KoreaMed CrossRef
  33. Sah S, Singh N, Singh R. 2017. Iron acquisition in maize (Zea mays L.) using Pseudomonas siderophore. 3 Biotech 7: 121.
    Pubmed KoreaMed CrossRef
  34. Shi P, Zhu K, Zhang Y, Chai T. 2016. Growth and cadmium accumulation of Solanum nigrum L. seedling were enhanced by heavy metal-tolerant strains of Pseudomonas aeruginosa. Water. Air. Soil Pollut. 227: 459.
    CrossRef
  35. Aloo BN, Makumba BA, Mbega ER. 2019. The potential of Bacilli rhizobacteria for sustainable crop production and environmental sustainability. Microbiol. Res. 219: 26-39.
    Pubmed CrossRef
  36. Sansinenea E, Ortiz A. 2011. Secondary metabolites of soil Bacillus spp. Biotechnol. Lett. 33: 1523-1538.
    Pubmed CrossRef
  37. Ghojavand H, Vahabzadeh F, Roayaei E, Shahraki AK. 2008. Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696). J. Colloid Interface Sci. 324: 172-176.
    Pubmed CrossRef
  38. Chandankere R, Yao J, Cai M, Masakorala K, Jain AK, Choi MMF. 2014. Properties and characterization of biosurfactant in crude oil biodegradation by bacterium Bacillus methylotrophicus USTBa. Fuel 122: 140-148.
    CrossRef
  39. Joshi SJ, Geetha SJ, Desai AJ. 2015. Characterization and application of biosurfactant produced by Bacillus licheniformis R2. Appl. Biochem. Biotechnol. 177: 346-361.
    Pubmed CrossRef
  40. Priya T, Usharani G. 2009. Comparative study for biosurfactant production by using Bacillus subtilis and Pseudomonas aeruginosa. Bot. Res. Int. 2: 284-287.
  41. Joshi SJ, Geetha SJ, Yadav S, Desai AJ. 2013. Optimization of bench-scale production of biosurfactant by Bacillus licheniformis R2. APCBEE Procedia 5: 232-236.
    CrossRef
  42. Mouafi FE, Abo Elsoud MM, Moharam ME. 2016. Optimization of biosurfactant production by Bacillus brevis using response surface methodology. Biotechnol. Rep. 9: 31-37.
    Pubmed KoreaMed CrossRef
  43. Saxena AK, Kumar M, Chakdar H, Anuroopa N, Bagyaraj DJ. 2020. Bacillus species in soil as a natural resource for plant health and nutrition. J. Appl. Microbiol. 128: 1583-1594.
    Pubmed CrossRef
  44. Mehta P, Walia A, Chauhan A, Kulshrestha S, Shirkot CK. 2013. Phosphate solubilisation and plant growth promoting potential by stress tolerant Bacillus sp. isolated from rhizosphere of apple orchards in trans Himalayan region of Himachal Pradesh. Ann. Appl. Biol. 163: 430-443.
    CrossRef
  45. Rahman CH, Miloud B, Rachid D, Ahcene B, Hakim H. 2017. Optimization of inorganic phosphate solubilization by Pseudomonas fluorescens and Bacillus sp. isolated from wheat rhizospheric soil. Int. J. Biosci. 10: 142-150.
    CrossRef
  46. Banerjee S, Palit R, Sengupta C, Standing D. 2010. Stress induced phosphate solubilization by 'Arthrobacter' sp. and 'Bacillus' sp. isolated from tomato rhizosphere. Aust. J. Crop Sci. 4: 378-383.
  47. Soni R, Keharia H. 2021. Phytostimulation and biocontrol potential of gram-positive endospore-forming Bacilli. Planta 254: 49.
    Pubmed CrossRef
  48. Yu S, Teng C, Bai X, Liang J, Song T, Dong L, Jin Y, Qu J. 2017. Optimization of siderophore production by Bacillus sp. PZ-1 and its potential enhancement of phytoextration of Pb from soil. J. Microbiol. Biotechnol. 27: 1500-1512.
    Pubmed CrossRef
  49. Yu X, Ai C, Xin L, Zhou G. 2011. The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur. J. Soil Biol. 47: 138-145.
    CrossRef
  50. Biedendieck R, Knuuti T, Moore SJ, Jahn D. 2021. The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology. Appl. Microbiol. Biotechnol. 105: 5719-5737.
    Pubmed KoreaMed CrossRef
  51. Bashir S, Iqbal A, Hasnain S, White JF. 2021. Screening of sunflower associated bacteria as biocontrol agents for plant growth promotion. Arch. Microbiol. 203: 4901-4912.
    Pubmed CrossRef
  52. Sharma K, Sharma S, Vaishnav A, Jain R, Singh D, Singh HB, et al. 2022. Salt-tolerant PGPR strain Priestia endophytica SK1 promotes fenugreek growth under salt stress by inducing nitrogen assimilation and secondary metabolites. J. Appl. Microbiol. 133: 2802-2813.
    Pubmed CrossRef
  53. Shahid M, Zeyad MT, Syed A, Singh UB, Mohamed A, Bahkali AH, et al. 2022. Stress-tolerant endophytic isolate Priestia aryabhattai BRP-9 modulates physio-biochemical mechanisms in wheat (Triticum aestivum L.) for enhanced salt tolerance. Int. J. Environ. Res. Public Health 19: 10883.
    Pubmed KoreaMed CrossRef
  54. Sandhu M, Paul AT, Proćków J, de la Lastra JMP, Jha PN. 2022. PCB-77 biodegradation potential of biosurfactant producing bacterial isolates recovered from contaminated soil. Front. Microbiol. 13: 952374.
    Pubmed KoreaMed CrossRef
  55. Siddiqui Z, Grohmann E, Malik A. 2023. Degradation of alkane hydrocarbons by Priestia megaterium ZS16 and sediments consortia with special reference to toxicity and oxidative stress induced by the sediments in the vicinity of an oil refinery. Chemosphere 317: 137886.
    Pubmed CrossRef
  56. Uyar E, Avcı T. 2023. Screening and molecular identification of biosurfactant/bioemulsifier producing bacteria from crude oil contaminated soils samples. Biologia 78: 2179-2193.
    CrossRef
  57. Kaminsky LM, Bell TH. 2022. Novel primers for quantification of Priestia megaterium populations in soil using qPCR. Appl. Soil Ecol. 180: 104628.
    CrossRef
  58. Hu X, Roberts DP, Xie L, Maul JE, Yu C, Li Y, et al. 2013. Development of a biologically based fertilizer, incorporating Bacillus megaterium A6, for improved phosphorus nutrition of oilseed rape. Can. J. Microbiol. 59: 231-236.
    Pubmed CrossRef
  59. Jiang H, Qi P, Wang T, Chi X, Wang M, Chen M, et al. 2019. Role of halotolerant phosphate-solubilising bacteria on growth promotion of peanut (Arachis hypogaea) under saline soil. Ann. Appl. Biol. 174: 20-30.
    CrossRef
  60. Lin XR, Chen HB, Li YX, Zhou ZH, Li JB, Wang YQ, et al. 2022. Priestia sp. LWS1 is a selenium-resistant plant growth-promoting bacterium that can enhance plant growth and selenium accumulation in Oryza sativa L. Agronomy 12: 1301.
    CrossRef
  61. Rehan M, Al-Turki A, Abdelmageed AHA, Abdelhameid NM, Omar AF. 2023. Performance of plant-growth-promoting rhizobacteria (PGPR) isolated from sandy soil on growth of tomato (Solanum lycopersicum L.). Plants 12: 1588.
    Pubmed KoreaMed CrossRef

Article Tools

Starts of Metrics

Share this article on :

Related articles in MBL

Most Searched Keywords ?

What is Most Searched Keywords?

  • It is most registrated keyword in articles at this journal during for 2 years.