The formation of bixbyite-type Mn2O3 via pyocyanin-dependent Mn(II) oxidation of soil-derived Pseudomonas aeruginosa

doi:10.21203/rs.3.rs-4015054/v1

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The formation of bixbyite-type Mn₂O₃ via pyocyanin-dependent Mn(II) oxidation of soil-derived Pseudomonas aeruginosa

https://doi.org/10.21203/rs.3.rs-4015054/v1

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Bacterial Mn(II) oxidation is believed to play a dominant role in accelerating the rate of Mn biomineralization in nature. Commonly, bacteria adopt two ways concerning Mn(II) oxidases and reactive oxygen species to oxidize Mn(II). In this study, a new strategy for bacterial Mn(II) oxidation involving the pyocyanin, a greenish blue phenazine pigment from Pseudomonas aeruginosa, was discovered. To begin with, a bacterial strain L3 was isolate from soils and identified as Pseudomonas aeruginosa, which exhibited the ability of Mn(II) oxidation. Next, the pyocyanin was purified from strain L3 cultures and proven to be involved in Mn(II) oxidation. Particularly, the oxidation of Mn(II) by pyocyanin was dependent on its ambient pH. In comparison with pH of 5 and 7, pyocyanin (the initial value of OD₃₈₇ was 0.56 at pH 2) showed a stronger capability of oxidizing Mn(II) at pH of 9, reaching 144.03 µg L^− 1 of Mn oxides after 108 h of Mn(II) oxidation, while pyocyanin ultimately produced 43.81 µg L^− 1 at pH of 7 and 3.32 µg L^− 1 at pH of 5, respectively. Further, strain L3 cultures were fractionated into three parts, i.e., the cell culture solution, fermentation supernatant, and cell suspension, and the Mn(II)-oxidizing activity was found to be distributed in the cell culture solution and fermentation supernatant, as evidenced by the formation of blackish glossy Mn oxides. Specifically, in the first half, the rate of Mn(II) oxidation by the fermentation supernatant was higher than that by the cell culture solution, whereas in the second half, the cell culture solution showed the much higher Mn(II)-oxidizing activity than did the fermentation supernatant. Last but not least, the collective results from mineral characterization demonstrated that, the Mn oxides produced by P. aeruginosa strain L3, either by the cell culture solution or by the fermentation supernatant, were bixbyite-type Mn₂O₃ with poor crystallinity.

Applied & Industrial Microbiology

Pseudomonas aeruginosa

pyocyanin

Mn(II) oxidation

bixbyite-type Mn2O3

Mn₂O₃ has been profoundly employed in a wide-ranging process of industrial manufacturing, such as energy storage, soft magnetic materials, rechargeable lithium ion batteries, gas sensors, molecular catalysis for carbon monoxide and nitrogen oxide from wastes (Chen et al., 2012; Parveen et al., 2022). Meanwhile, in the field of clinical diagnosis, Mn₂O₃ is increasingly tested to construct electrochemical biosensors for substrates detection (Wang et al., 2017; Sun et al., 2021). To date, a number of well-developed chemical procedures have been used to synthesize Mn₂O₃, comprising hydrothermal, electrospinning, solvothermal, wet chemical, and various precipitation methods (Parveen et al., 2022; Ansari et al., 2020). In addition to these chemical ways, biological synthesis for Mn oxides recently receives popular attention because of their generally high catalytic reactivity, and also the necessity of good biocompatible materials in industries like environment, health, and medicine (Hoseinpour and Ghaemi, 2018; Remucal and Ginder-Vogel, 2014).

Biological synthesis of Mn oxides essentially refers to the transformation of soluble Mn(II) to insoluble Mn(III/IV) minerals by organisms such as bacteria, fungi and plant extracts (Hoseinpour and Ghaemi, 2018; Learman et al., 2011). Particularly, bacterial Mn(II) oxidation has been extensively reported, exhibiting a high efficient potential of synthesizing Mn oxides, and also a great environment implication in global biogeochemical cycling (Yu and Leadbetter, 2020; Mirna et al., 2019; Chen et al., 2020). Mn(II) oxidation occurs in a wide variety of bacterial species, covering α, β, γ-Proteobacteria, Firmicutes, and Actinobacteria (Tebo et al., 2005). Generally, these bacterial species may employ four different types of strategies to oxidize Mn(II) as follows: (i) direct oxidation of Mn(II) by the multicopper oxidases (MCOs), which share low homology except for their copper binding motifs (Ridge et al., 2007); (ii) direct oxidation of Mn(II) by the peroxidase cyclooxygenases, which feature animal heme peroxidase domains and hemolysin-type Ca(II) binding domains (Geszvain et al., 2016; Nakama et al., 2014); (iii) light-driven anaerobic microbial oxidation of Mn(II) (Mirna et al., 2019); (iv) indirect oxidation of Mn(II) by superoxide coupled to hydrogen peroxide consumption (Chen et al., 2022; Andeer et al., 2015).

Pseudomonas aeruginosa is a Gram-negative bacterium that mostly inhabits in clinical settings, capable of causing a wide array of life-threatening acute and chronic infections, particularly in patients with cancer, post-surgery, severe burns or infected by human immunodeficiency virus (HIV) (Moradali, et al., 2017). Frequently, common antibiotics show limited efficacy due to high intrinsic resistance and adaptability of P. aeruginosa (Pang et al., 2019). Several other defensive strategies are also harnessed by P. aeruginosa to protect themselves from surrounding environmental stresses, including biofilm formation, quorum sensing, motility-sessility switch, and the CRISPR-Cas systems (Thi et al., 2020; Moradali, et al., 2017).

Recent studies have shown that bacterial strains within P. aeruginosa also inhabit the heavy metal contaminated soils, exhibiting environment-dependent physiological features, such as the ability to absorb or oxidize metal ions (Li et al., 2023; Ren et al., 2023; Singh, et al., 2013; Silva et al., 2009). Specifically, regarding the Mn(II) oxidation occurred in P. aeruginosa, at the time of writing, there are only two reports can be found after exhaustingly searching literatures in web of science (Li et al., 2023; Ren et al., 2023). Ren et al. (2023) speculated that Pseudomonas aeruginosa MQ2 might make use of its two MCOs and a manganese-containing superoxide dismutase (MnSOD) to directly oxidize Mn(II) ions, and also suggested that reactive oxygen species (ROS) could enhance the rate of Mn(II) oxidation. The Mn oxides produced by P. aeruginosa MQ2 were characterized as the mixture of MnO₂, Mn₃O₄ and MnCO₃ (Ren et al., 2023). In parallel, Li et al. (2023) reported that the Mn oxides generated by another Pseudomonas aeruginosa strain, PA-1 (isolated from the manganese mining area soils), exhibited three characteristic peaks of X-ray diffraction at 2θ = 12.0°, 36°, and 65°, which could be assigned to δ-MnO₂ (birnessite-like Mn oxides). Yet, the authors did not explicitly interpret how P. aeruginosa PA-1 oxidized Mn(II) ions (Li et al., 2023).

As a typical pigment-producing bacterial species, the most of P. aeruginosa strains (90%~95%) are able to produce pyocyanin, a greenish blue phenazine pigment (Abdelaziz et al., 2023). It is a redox-active metabolite that can gain and lose electrons reversibly under physiological conditions (Perry et al., 2022). Owing to this, pyocyanin possesses many biological activities, e.g., antibacterial, antifungal, anti-quorum sensing, antioxidant, anti-inflammatory and etc. Moreover, it can be used in areas like agriculture and aquaculture, as wells as a stable colorant agent, an electron shuttle in microbial fuel cells, and so on (Abdelaziz et al., 2023). Nevertheless, to our knowledge, the role of pyocyanin in Mn(II) oxidation has never been concerned previously. Whether it could be involved in Mn(II) oxidation by P. aeruginosa remains to be investigated.

To enrich the knowledge of Mn(II) oxidation by P. aeruginosa, particularly concerning the mode of oxidation and the type of Mn oxides, which are different from that of the strains i.e. MQ2 and PA-1 as described above, we herein introduce a newly found Pseudomonas aeruginosa strain L3 from heavy metal contaminated soils, to illustrate that this bacterium is a pyocyanin-dependent producer of bixbyite-type Mn₂O₃.

2.1 Isolation of bacterial strain L3

The strain L3 was originally isolated from the surface layer of heavy metal contaminated soils (25 cm) in the vicinity of a ceased smelting factory in Zhuzhou (113°09′11.48′′E and 27°87′07.41′′N), Hunan Province, PR China. The soils consisted of 32.33 ± 1.01% sand, 41.17 ± 1.12% silt, and 26.50 ± 1.73% clay, which was determined as clay loam according to the hydrometer method (Gee and Bauder, 1986; Li et al., 2020). The soil properties were listed as follows: pH 6.62–7.08, 71.7 mmol kg^− 1 of cation exchange capacity (CEC), 3.18 ± 0.15% of organic matter, and the concentrations of several common heavy metals were 1140 ± 70 mg kg^− 1 of Pb, 87 ± 5 mg kg^− 1 of Cr, 70 ± 3 mg kg^− 1 of Cd, 437 ± 14 mg kg^− 1 of Cu, 815 ± 21 mg kg^− 1 of Mn, and 1760 ± 98 mg kg^− 1 of Zn, respectively, which were far beyond the risk values for soil environmental quality of China (GB15618-2018). The strain L3 was obtained by using a gradient dilution method as previously described by Li et al. (2020). Briefly, soil samples were sufficiently mixed with sterile ddH₂O at a ratio of 1:9 (w/v) in an Erlenmeyer flask. The soil suspension was then serially diluted with sterile ddH₂O at the same ratio, and spread onto LB agar plates supplemented with 50 mM of Mn(II) for incubation for 72 h at 35℃. A single Mn(II)-tolerant bacterial colony was selected to streak on Luria-Bertani (LB) agar plates (Chen et al., 2022) for purification several times, and was finally denoted as L3.

2.2 Phylogenetic identification of strain L3

The genomic DNA of strain L3, prepared from a pure culture using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech), was used as the PCR template to amplify the 16S rRNA gene, via a pair of universal primers, 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ) (Weisburg et al., 1991). The sequenced amplification fragments of 16S rRNA gene were subject to phylogenetic identification by using the BLAST search program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and MEGA X software (Li et al., 2022). Briefly, the phylogenetic tree was constructed by employing the neighbour-joining (NJ) method in MEGA X software to analyze the evolutionary relationship between strain L3 and close bacterial species. Evolutionary distances were calculated by Kimura’s two-parameter and Gamma distributed with invariant sites models. Based on 1000 replicates, bootstrap analysis was conducted to exhibit confidence levels for branches (Li et al., 2022).

2.3 Microscopic observation of strain L3

The scanning electron microscope (SEM) was used to observe the cell morphology of strain L3 (ZEISS EVO MA 10, Carl Zeiss AG). Bacterial cells grown in LB liquid medium were collected by centrifuging at 12,000 rpm for 3 min under 4℃. The cell samples were treated with 2.5% glutaraldehyde solution for 12 h, and then were rinsed with 0.1 M phosphate buffer (pH 7.0) three times (each time for 15 min). Subsequently, the cell samples were dehydrated by ethanol solutions with a concentration gradient of 30%, 50%, 70%, 80%, 90%, 95% and 100% (each concentration for 20 min). After that, cell samples were treated with the mixed solution of ethanol and iso-amyl acetate (v:v = 1:1) for 30 min, and then were treated with pure iso-amyl acetate for 1 h, prior to drying at critical point and coating.

2.4 Determination of growth and Mn(II) oxidation for strain L3

The strain L3 was incubated in the sterile LB liquid medium for cell growth with a condition of darkness, 180 rpm and 35°C, which were maintained in all experiments unless otherwise indicated. The growth of cells was monitored via optical density (OD) at 600 nm using an ultraviolet-visible (UV-Vis) spectrophotometer (TU-1810, Pgeneral, China), and the pH value of culture solution was continuously monitored for variation during the cell growth.

Leucoberbelin Blue (LBB) was used to evaluate the ability of strain L3 to oxidize Mn(II) (Li et al., 2020). In brief, the cells incubated in LB liquid medium (supplemented with Mn(II) ions) were mixed with 0.04% LBB (prepared by 45 mM acetic acid) at a volume ratio of 1:5 to see if the mixture would become blue (Krumbein and Altmann, 1973).

2.5 Pyocyanin extraction and its Mn(II) oxidation determination

The procedure for the extraction of pyocyanin was moderately modified from the methods as describe by Abdelaziz et al. (2023). Briefly, 25 mL of cell culture solution was sufficiently mixed with 15 mL of chloroform for 5 min, which was then centrifuged at 8,000 rpm for 10 min. The supernatant was discarded, and 10 mL of 0.2 M HCl was subsequently added into the remaining solution, sufficiently mixing for 5 min. After that, the remaining solution was centrifuged at 8,000 rpm for 10 min, and the supernatant was the final solution of pyocyanin. The extracted pyocyanin was monitored by using the UV-Vis spectrophotometer (TU-1810, Pgeneral) in a range of 200–600 nm.

LBB staining was carried out to assess whether the pyocyanin was involved in Mn(II) oxidation. Prior to staining, the pH of the pyocyanin solution was adjusted to be acidic (pH of 5), neutral (pH of 7), and alkaline (pH of 9), respectively. Then, these pyocyanin solutions with different pH were supplemented with 25 mM of Mn(II) ions to incubate for 3 d with 180 rpm at 35℃.

Quantification of Mn oxides produced by pyocyanin was further colorimetrically performed through LBB staining as described previously (Wright et al., 2018; Boogerd and de Vrind, 1987; Krumbein and Altmann, 1973). In brief, known concentrations of KMnO₄ were used to plot the standard curve of 10–200 µg L^− 1. The absorbance values of samples were visualized spectrophotometrically at 623 nm (Multiscan MK3, Thermo). It was assumed that all Mn oxides were MnO₂ or Mn₂O₃ when calculating Mn(III,IV) oxide concentrations (Tebo et al., 2007).

The concentrations of Mn oxides were calculated by the following equation:

Y = 5/2·(kA + b) (1)

Where Y is the concentration of Mn oxides; A is the absorbance of samples; k is slope of the standard curve; b is y-axis intercept of the standard curve. 5/2 is transformation coefficient of concentrations between KMnO₄ and MnO₂ (or Mn₂O₃).

2.6 Purification of Mn oxides produced by strain L3

Mn oxides were collected from strain L3 through two ways, i.e., (i) purifying Mn oxides from cell culture solution (containing cells), and (ii) purifying them from the supernatant after centrifuging, filtering (0.22 µm membrane) the cell culture solution, which did not contain cells and was denoted as the fermentation supernatant hereinafter. Before purifying Mn oxides, the cell culture solution and the fermentation supernatant were exposed to 25 mM of Mn(II) for 5 d, shaken at 35℃ with 180 rpm, respectively. After that, the purification of Mn oxides from these solutions was carried out according to the procedure as previously described by Luo et al. (2022) that exhibited chemical inertness towards the structure of Mn oxides.

Briefly, the solution samples were centrifuged at 4,000 rpm for 10 min at room temperature, discarding the supernatant, and were sonicated in phenol: chloroform (v:v = 1:1) for 30 min (step 1). Then, the suspension of step 1 was added with methanol: chloroform: ddH₂O (v:v:v = 12:5:3) to further sonicate for 10 min (step 2). Next, the suspension of step 2 was centrifuged to obtain precipitates, which were acidized to pH 3 by HCl and shaken at 180 rpm for 15 min (step 3). The suspension of step 3 was centrifuged to obtain the remaining precipitates, which were subsequently shaken in 0.17% NaClO for 30 min (step 4). Finally, the suspension of step 4 was centrifuged to obtain Mn oxides, which were rinsed with ddH₂O and dried at 60°C for 1 h.

The procedure for quantification of Mn oxides produced by the cell culture solution and fermentation supernatant was in agreement with the description of section 2.5, except for repreparation of the standard curve of 10-1500 µg L^− 1 and removal of the cells in samples before absorbance measurement.

2.7 Mineral Characterization

The SEM equipped with Energy dispersive X-ray spectrometer (EDS) was used to record the morphology of purified Mn oxides from strain L3 and their element composition (ZEISS EVO MA 10, Carl Zeiss AG). Transmission electron microscopy (TEM) equipped with selected area electron diffraction (SAED) was employed to observe the crystal lattice features of Mn oxides (Tecnai G2 F20, FEI). X-ray diffractometer (Bruker D8 Advance) was performed to test the patterns of X-ray diffraction (XRD) for Mn oxides over the 2θ range of 10–80°, which were further matched with the standard powder diffraction files (PDF) by MDI Jade 6 to fully understand the crystal phase of Mn oxides. Fourier Transform infrared spectroscopy (FT-IR) spectra were recorded to analyze the functional groups on the surface of Mn oxides by using a PerkinElmer spectrometer (L160000V). The powder samples of Mn oxides were encapsulated by KBr at a mass ratio of 1:100 (m_{Mn oxides}: m_KBr), scanning in the range of 500–4000 cm^− 1. X-ray photoelectron spectroscopy (XPS) was performed to investigate the chemical valence state of Mn and O on the surface of Mn oxides by using a Thermo Scientific spectrometer (K-Alpha). The specific surface area (SSA) and pore size of Mn oxides were detected by an Autosorb-1 physical adsorption analyzer (Quantachrome, USA), according to the Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods.

3.1 Identification of bacterial strain L3

The comparison of 16S rRNA gene sequences is a reliable way to evaluate the affiliation of bacterial species. The similarity of 16S rRNA gene sequences higher than 97% between bacterial strains could be considered as a generally accepted criterion to delineate them as the same species (Stackebrandt and Goebel, 1994). After sequencing the amplified fragments obtained from the universal primers of 27F and 1492R, 1528 bp complete sequence of 16S rRNA gene from strain L3 was deposited at Genbank with the accession number of ON909190. Through the alignment searching for this complete 16S rRNA gene sequence in the BLAST program, strain L3 exhibited a 100% similarity to numerous members of Pseudomonas aeruginosa, indicating the affiliation of strain L3 to P. aeruginosa. A phylogenetic tree was presented to illustrate an evolutionary relationship between strain L3 and its highly similar neighbors within the genus Pseudomonas (Fig. 1A). The 100% bootstrap value on the branch that consisted of strain L3 and another two strains in P. aeruginosa, indicated that the clade topology around strain L3 was sufficiently supported, evolutionarily suggesting the membership of strain L3 within P. aeruginosa.

The SEM image of P. aeruginosa L3 displayed that the cells were rod-shaped with the dimensions of ~ 0.4×1.5 µm (width×length) (Fig. 1B), essentially in agreement with the previous typical description for the species of P. aeruginosa (Horst, et al., 2010). As shown in Fig. 1C, the growth of strain L3 occurred exponentially in the first 30 h, and then reached to a plateau since the 36th h. After 52 h of incubation, the growth curve began to fall, indicating that cells underwent a transition from the stationary phase to the recession phase. Meanwhile, the pH variation was recorded in Fig. 1D. As the incubation proceeded, the culture solution of cells displayed a rising trend from pH of 7 within 56 h, gradually reaching to a plateau of pH 9.3 since the 60th h. Such an increasing trend for pH could be attributed to the alkaline metabolites produced constantly in cell metabolism (Luo et al., 2022; LaBauve and Wargo, 2012).

As illustrated in Fig. 1E, the oxidation ability of strain L3 for Mn(II) was preliminarily demonstrated by LBB staining assays (Krumbein and Altmann, 1973). It can be seen that after 52 h of incubation, the cells which were cultured in LB liquid medium containing 5 or 6 mM of Mn(II), made the reagent of 0.04% LBB stain blue color, indicating the presence of Mn oxides around cells (Chen et al., 2022). By contrast, in the case of Mn(II) addition ranging from 0 to 4 mM, no blue staining evidently occurred. These results suggest that, when employing the LBB assay to test the Mn(II) oxidation of strain L3, 5 mM of Mn(II) was the limit of detection for the LBB staining test. Besides, the growth curves corresponding to these 0–6 mM Mn(II) concentrations, showed that the addition of Mn(II) did not markedly retard the cell growth within 12 h, but significantly inhibited the cell proliferation after 12 h of incubation. This is presumably that, on the one hand, Mn(II) exposure at a period of time will be toxic to bacterial cells via energy metabolism deficiency (Kaur et al., 2017), and on the other hand, since P. aeruginosa is a well-known species that readily forms biofilm when encountering ambient stress, the cellular aggregation in culture solution may also result in the decline of OD values (Thi et al., 2020).

3.2 Growth of strain L3 on LB agar plates at different Mn(II) concentrations

The growth of strain L3 was examined on LB agar plates supplemented with different Mn(II) concentrations ranging from 0 to 80 mM. As shown in Fig. 2, compared to the control group (0 mM), the presence of Mn(II) in the plates resulted in the growth inhibition. In the range of 5–20 mM Mn(II), it can be seen that the streaked colonies were somewhat dehydrated to form a crust. As the concentration increased to 25 mM, the streaked colonies exhibited green. In the range of 30–80 mM Mn(II), the growth of colonies was significantly suppressed, and the maximum concentration that L3 can tolerate was considered as 50 mM, as evidenced by sporadic colonies occurred in the plate (Fig. 2).

Here, the most noteworthy phenomenon is that at 25 mM of Mn(II), this concentration of pressure largely stimulated the formation of greenish biofilm. Biofilm, consisted of extracellular polymeric substances (EPS) and bacterial cells, can help cells effectively withstand the environmental stress by creating comfortable living conditions for them (Flemming et al., 2007). For P. aeruginosa, pyocyanin plays a key role in the formation of greenish biofilm, since the pyocyanin-extracellular DNA complex interferes with the hydrophobicity of cells and advances the development of robust biofilms (Das et al., 2013). Moreover, pyocyanin can enhance the resistance of P. aeruginosa to metal ions by controlling the genes responsible for efflux pumps (Muller and Merrett, 2014).

3.3 Pyocyanin was involved in Mn(II) oxidation

To exactly investigate whether pyocyanin would be involved in Mn(II) oxidation, it was firstly extracted from the cell culture solution and examined by UV-vis spectrophotometer for its characteristic peaks. As shown in Fig. 3A, the initially extracted pyocyanin (pH = 2) exhibited two characteristic peaks at 278 and 387 nm in the range of 200–600 nm. When the pH was increased from 2 to 9, the characteristic peak on the left shifted from 278 to 311 nm, while the one on the right remained at 387 nm, suggesting that the right peak at 387 nm can be used as a stable characteristic one for pyocyanin detection in the pH range of 2–9. These results were essentially in agreement with the description of pyocyanin characteristic peaks for Pseudomonas aeruginosa P32, in which the extracted pyocyanin showed that the peaks occurred at 263 and 367 nm (Abdelaziz, et al., 2022).

It can be seen from Fig. 3B that the production of pyocyanin by strain L3 mainly occurred in the first 12 h of cell growth, and since then, the content of pyocyanin was not increased as cell proliferation any more. Also, the exposure of Mn(II) ions to cells had a negative effect on the yield of pyocyanin. As illustrated in Fig. 3C, in the absence of Mn(II), the maximum ratio of OD₃₈₇ to OD₆₀₀, which can reflect the capability of cells to yield pyocyanin, reached 0.75 at the 9th h. By comparison, in the presence of Mn(II), the maximum ratios of OD₃₈₇ to OD₆₀₀ gradually declined as Mn(II) concentrations, suggesting that the higher the Mn(II) concentration was, the fewer the yield of pyocyanin on a unit number of cells was. Moreover, Fig. 3C showed that, there was an obvious time lag for occurrence of the maximum ratio of OD₃₈₇ to OD₆₀₀ between the cells without Mn(II) exposure and the ones with Mn(II) exposure, as reflected by the earlier fade of green color at the higher Mn(II) concentration in Fig. 3D.

The oxidation of Mn(II) by pyocyanin was dependent on the ambient pH. Under the neutral (pH = 7) and alkaline (pH = 9) conditions, pyocyanin displayed green and blue, respectively, both of which showed the ability to oxidize Mn(II), as qualitatively evidenced by the formation of yellowish-brown particles and the positive staining of LBB (Fig. 3E). However, the acidic environment (pH = 5) was not in favor of Mn(II) oxidation by pyocyanin (Fig. 3E).

Further, pyocyanin was quantitatively analyzed for its capability of oxidizing Mn(II) in different pH environments. As shown in Fig. 3F, compared to pH of 5 and 7, pyocyanin exhibited the strongest capability of oxidizing Mn(II) at pH of 9, reaching 144.03 µg L^− 1 of Mn oxides after 108 h of Mn(II) oxidation. At pH of 7, pyocyanin finally generated 43.81 µg L^− 1 of Mn oxides through oxidation. In sharp contrast, only 3.32 µg L^− 1 of Mn oxides were produced by pyocyanin at pH of 5. This enhanced capability of pyocyanin for Mn(II) oxidation as pH value, was presumably attributed to the redox state of pyocyanin. At the acidic condition, pyocyanin was readily in the reduced state upon gaining two ambient protons/electrons (Perry et al., 2022). Thus, most of it may no longer accept the protons/electrons from Mn(II), leading to the low yield of Mn oxides. As the pH increased, particularly in alkaline solution, most of it was commonly in the oxidized state, which was ready for Mn(II) oxidation.

3.4 Distribution of Mn(II)-oxidizing activity in fractionated cultures

The L3 cultures were fractionated into three parts, i.e., cell culture solution, fermentation supernatant, and cell suspension, as illustrated in Fig. 4A. The Mn(II)-oxidizing activity was distributed both in the cell culture solution and fermentation supernatant, as evidenced by the glossy black Mn oxides purified from them, respectively (Fig. 4A). For the cell suspension, its Mn(II)-oxidizing activity was insufficiently supported for the purification of Mn oxides.

The Mn(II)-oxidizing activity was compared between the cell culture solution and fermentation supernatant. Within 40 h, the rate of Mn(II) oxidation by the fermentation supernatant was higher than that by the cell culture solution. After then, on the contrary, the cell culture solution showed the much higher Mn(II)-oxidizing activity than did the fermentation supernatant, ultimately reaching 1.17 mg L^− 1 of Mn oxides versus 0.44 mg L^− 1 of Mn oxides obtained from the fermentation supernatant (Fig. 4B).

Overall, Mn oxides experienced a linear growth in the fermentation supernatant over the course of 80 h. This is presumably because the total content of pyocyanin, the major substrate responsible for Mn(II) oxidation in the supernatant, was constant. The phenomenon that the growth of Mn oxides in the cell culture solution was slower than that in the fermentation supernatant in the first 40 h, may largely be due to the fact that Mn(II) inhibited the excretion of pyocyanin from cells (Fig. 3C). Additionally, the presence of Mn(II), at this stage, may play a negative role in cell viability via energy metabolism deficiency (Kaur et al., 2017). Interestingly, Emerson and Ghiorse (1992) also reported a similar reproducible phenomenon for Leptothrix discophora SP-6(sl), in which the supernatants after centrifugation were approximately 4-fold higher active than the complete cultures in Mn(II) oxidation. They speculated that the presence of SP-6(sl) cells could have inhibited the Mn(II)-oxidizing activity in the medium, but the mechanism of such inhibition was unexplained (Emerson and Ghiorse, 1992).

The reasons for the superiority of cell culture solution to fermentation supernatant in Mn(II) oxidation in the following 40 h, could possibly be explained as follows. Despite the fact that the presence of Mn(II) had a negative effect on the cell growth at the early stage, the total amounts of cells, including newly born, recessionary, dead and even lytic cells, were gradually increasing over the period of 80 h. These constituents may result in the higher total amount of pyocyanin in the cell culture solution than that in the fermentation supernatant at the late stage. The Mn(II)-oxidizing enzymes in cells may also facilitate Mn(II) oxidation (Ren et al., 2023; Li et al., 2020). Enzymatic Mn(II) oxidation commonly needs a relatively long time, because intracellular Mn(II)-oxidizing proteins are believed to be transported out the membrane to fulfil the Mn(II) oxidation (Li et al., 2020). Furthermore, an experimental factor that cannot be ignored was that the pH of cell culture solution increased as time, which was in favor of LBB staining.

3.5 Characterization of Mn oxides produced by strain L3

The Mn oxides purified from the cell culture solution (denoted as Mn oxides-C) and the fermentation supernatant (denoted as Mn oxides-F) as indicated in Fig. 4A were characterized, respectively. The results of SEM-EDS (Fig. 5A-F) showed that, the two kinds of Mn oxides were both in the size range of 40–80 µm with the slippery surfaces, and they were primarily composed of four elements comprising Mn, O, C, and N, suggesting the presence of impurities in addition to Mn and O, as observed previously in other biogenic Mn oxides (Luo et al., 2022; Tran et al., 2018). Generally, biogenic Mn oxides featured their high SSAs in previous studies, such as 49 m²/g of bixbyite-like Mn₂O₃ produced by Bacillus CUA (Zhang et al., 2014), 98 m²/g of δ-MnO₂ produced by Pseudomonas putida MnB1 (Villalobos et al., 2003), and 224 m²/g of Mn oxides produced by Leptothrix discophora SS-1 (Nelson et al., 1999). However, in this study, the SSA of Mn oxides produced by P. aeruginosa L3 was much lower than that of Mn oxides generated by strains mentioned above, only ranging from 0.6675 to 0.6784 m²/g (Table 1). Actually, the blackish glossy surface of Mn oxides as shown in Fig. 4A can also mirror the results of low SSA for P. aeruginosa L3.

Table 1

The BET-specific surface areas and pore parameters of Mn oxides.
Sample	BET-specific surface areas (m²/g)	Total pore volume (cm³/g)	Average pore diameter (nm)
Mn oxides purified from the culture solution of strain L3	0.6675	0.003247	17.9522
Mn oxides purified from the fermentation supernatant of strain L3	0.6784	0.002830	19.6408

TEM-SAED imaging revealed that the both Mn oxides were poor in crystallinity and did not sufficiently exhibit lattice fringes, as evidenced by their dim polycrystalline diffraction rings (Fig. 5G-J). Nevertheless, the diffraction rings of (921), (521) and (321) from the Mn oxides-C (Fig. 5H), and the diffraction rings of (822), (611) and (222) from the Mn oxides-F (Fig. 5J), were all attributed to the crystal planes of bixbyite-type Mn₂O₃ (α-Mn₂O₃).

XRD tests were performed to reconfirm the crystalline features of bixbyite-type Mn₂O₃ produced by L3. As shown in Fig. 6A, four characteristic peaks at 2θ = 23.132°, 32.952°, 55.191° and 65.807° were found for the both biogenic Mn oxides, corresponding to the crystal planes of (211), (222), (440) and (622) of bixbyite-type Mn₂O₃ (PDF#00-041-1442), respectively. Nonetheless, the noisy signals of low intensities for these characteristic peaks suggested the poor crystallinity of the two biogenic Mn oxides. Thus, the Mn oxides produced by L3 can be eventually assigned to bixbyite-type Mn₂O₃ with a poor crystallinity.

The FT-IR spectra of Mn oxides-C and Mn oxides-F were compared for the difference of functional groups on their surfaces. Except for the characteristic peaks at 1542, 1401 and 560 cm^− 1, the other characteristic peaks of Mn oxides-F were essentially shared by Mn oxides-C (Fig. 6B). For instance, the characteristic peak at 3416 cm^− 1 of Mn oxides-F, which indicated the stretching vibration of hydroxyl groups accompanied with the bending vibration of adsorbed water, was nearly equivalent to 3414 cm^− 1 of Mn oxides-C. The characteristic peaks of 2924 and 2925 cm^− 1 for Mn oxides-C and Mn-oxides-F, respectively, suggested the C-H bending vibration (Khan et al., 2015). The peak at 1638 cm^− 1 possibly indicated the presence of hydroxyl groups and amide linkages in the two Mn oxides (Li et al., 2020; Khan et al., 2015). The peak at 1114 cm^− 1 shared by the two Mn oxides probably indicated the stretching vibration of C-O (Gao et al., 2022). The major differences in FT-IR spectra between the two Mn oxides occurred at 1542, 1401 and 560 cm^− 1. Specifically, the peaks at 1542 and 1401 cm^− 1 which were present in Mn oxides-F but absent in Mn oxides-C, presumably suggested that Mn oxides-F were richer in amide linkages, carboxyl and hydroxyl groups than Mn oxides-C (Gao et al., 2022). Additionally, albeit the peak at 588 cm^− 1 shifted from Mn oxides-C to 560 cm^− 1 for Mn oxides-F, these peaks in the range of 500–700 cm^− 1 remained the lattice vibration of Mn-O bond (Gillot et al., 2001; Liu and Ooi, 2003).

XPS analysis was further performed to investigate the form of Mn and O on the surface of Mn oxides-C and Mn oxides-F. The Mn 2p spectra of Mn oxides-C showed that peak positions of Mn(III) and Mn(IV) were 641.7 and 643.3 eV at a Mn2p_3/2 level, and 653.3 and 655.1 eV at a Mn2p_1/2 level, respectively (Fig. 6C). The Mn 2p spectra of Mn oxides-F showed that peak positions of Mn(III) and Mn(IV) were 641.7 and 643.6 eV at a Mn2p_3/2 level, and 653.5 and 655.3 eV at a Mn2p_1/2 level, respectively (Fig. 6D). The percents of Mn(III) and Mn(IV) for Mn oxides-C were 64.03% and 35.97%, respectively, while the percents of Mn(III) and Mn(IV) for Mn oxides-F were 54.07% and 45.93%, respectively (Fig. 6C and D). The spectra of O 1s showed that, two peaks were found at 531.8 and 533.1 eV for oxides-C (corresponding to the hydroxyl oxygen species and the oxygen species in the water molecule, respectively), while the counterparts for oxides-F were found at 531.5 and 533.0 eV (Fig. 6E and F). These results indicated that, the percents of hydroxyl and water O on the surface of Mn oxides-C were 73.2% and 26.8%, respectively, while that of Mn oxides-F were 79.59% and 20.41%, respectively (Fig. 6E and F).

In general, environmental Mn(II)-oxidizing bacteria, including marine and freshwater, and terrestrial soil-derived ones, were mostly reported to produce birnessite (δ-MnO₂-like phyllo-manganate) via Mn(II) oxidation (Luo et al., 2022). So far, only a few soil-derived Mn(II)-oxidizing bacteria were reported to produce bixbyite-type Mn₂O₃, such as Bacillus CUA (Zhang et al., 2014), Mesorhizobium australicum T-G1 (Bohu et al., 2015), Bacillus mycoides BM228 (Jeyaraj and Subramanian, 2022), and Providencia manganoxydans LLDRA6 (Li et al., 2022; Luo et al., 2022). Based on the above results of mineral characterization, we have demonstrated that P. aeruginosa L3 is a new member of soil-originated producers that are able to synthesize bixbyite-type Mn₂O₃.

In this work, a novel bacterial Mn(II) oxidation pattern was discovered in a soil-derived Pseudomonas aeruginosa strain L3. Unlike the conventional Mn(II) oxidation strategies involving Mn(II) oxidases (direct oxidation) and ROS (indirect oxidation), this strain employed a secondary metabolite, namely pyocyanin, to oxidize Mn(II). Pyocyanin exhibited a higher capability of oxidizing Mn(II) under the alkaline condition in comparison to acidic and neutral conditions. This was because it was commonly in the oxidized state, readily gaining two protons/electrons from Mn(II). Besides, the blackish glossy Mn oxides can be purified from the cell culture solution and the fermentation supernatant of strain L3, respectively. Mineral characterization results suggested that these blackish glossy Mn oxides were poorly crystalline bixbyite-type Mn₂O₃. Taken together, discovery of this new strategy of Mn(II) oxidation by pyocyanin benefits to advance the understanding of non-enzymatic (indirect) Mn(II) oxidation in addition to ROS.

Funding

This research was supported by the Key Research and Development Program of Hunan Province (2023NK2024), the National Natural Science Foundation of China (32171622 and 51774129), and the Science and Technology Innovation Program of Hunan Province (2022RC1128, 2023NK4295 and 2023JJ50169).

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The 16S rRNA gene sequence of Pseudomonas aeruginosa L3 included in this study has been deposited at the GenBank with the accession number ON909190.

Author Contributions

DL conceived and designed the study. LX and FL retrieved the literature and drafted the manuscript. FT, SC, MH, LQ, YG, and HS participated in the study design and editing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Dr. Ding Li sincerely thanks his classmates of Party School of the Hunan Committee of C.P.C. (Hunan academy of Governance), Lei Zhao, Ping Xiao, and Xin He for their constructive comments on the manuscript editing.

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The authors declare no competing interests.

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The formation of bixbyite-type Mn₂O₃ via pyocyanin-dependent Mn(II) oxidation of soil-derived Pseudomonas aeruginosa

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Isolation of bacterial strain L3

2.2 Phylogenetic identification of strain L3

2.3 Microscopic observation of strain L3

2.4 Determination of growth and Mn(II) oxidation for strain L3

2.5 Pyocyanin extraction and its Mn(II) oxidation determination

2.6 Purification of Mn oxides produced by strain L3

2.7 Mineral Characterization

3 Results and discussion

3.1 Identification of bacterial strain L3

3.2 Growth of strain L3 on LB agar plates at different Mn(II) concentrations

3.3 Pyocyanin was involved in Mn(II) oxidation

3.4 Distribution of Mn(II)-oxidizing activity in fractionated cultures

3.5 Characterization of Mn oxides produced by strain L3

Conclusion

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