A Novel Selenite-Reducing Bacterium Bacillus pseudomycoides SA14 Isolated from Se-Enriched Soil and Its Potential Se Biofortification on Brassica chinensis L.

Abstract

Microbial participation in biofortification can improve the availability of selenium (Se) in soil and contribute to the enrichment of Se in crops. In this study, a selenite (Se(IV)) reducing strain was isolated from Se-rich soil, and its Se transformation and bio-enhancement ability were studied. The strain was identified as Bacillus pseudomycoides and could reduce more than 93.48% of 1.0 mM Se(IV) in 54 h. The results of scanning electron microscope (SEM) and energy dispersive X-ray spectrometry (EDS) showed that Se(IV) was reduced to Se(0), and Se nanoparticles (SeNPs) were eventually formed. In pot experiments, B. pseudomycoides SA14 could promote the bioavailable Se in soils and the concentration of Se in Brassica chinensis L.. The concentrations of water-soluble Se, ion exchange Se and carbonate-binding Se in soil were increased by 23.13%, 22.05% and 30.89%, respectively. The Se concentration of Brassica chinensis L. in pot experiments was increased by 145.05%. The relative abundance of Bacillus in soil increased from 0.97% to 2.08% in the pot experiments. As far as we know, this is the first report of Se reduction by B. pseudomycoides. This study might provide a prospective strategy for microbial fortification of Se in crops.

Graphical abstract

Keywords

Se(IV) reduction / Bacillus pseudomycoides / bioavailability / biofortification / dominant community / natural environment

0 INTRODUCTION

Selenium (Se) is an essential trace element for life (Zhao et al., 2020; Chauhan et al., 2019; El-Ramady et al., 2015). Insufficient Se intake by humans will lead to some endemic epidemics, such as Keshan disease and Kashin-Beck disease, whereas excessive Se intake can easily lead to selenosis (Wang Q et al., 2022; Wang X et al., 2019; Zhang X et al., 2019). Generally, Se is absorbed by humans through eating Se-enriched plants and crops, which absorb and transfer Se from soils (D’Amato et al., 2020). Previous studies have shown that Se-rich soils are generally distributed in spots rather than flakes, and Se deficiency in soils is a global problem (Araújo do Nascimento et al., 2021; Zhong et al., 2021). In China, 72% of areas are Se-deficient, whereas the levels of Se in soils in Jinhua City of Zhejiang Province, Guizhou Province, Enshi City of Hubei Province, and parts of southwestern Shanxi Province are higher than the global average (0.40 mg/kg) (Li et al., 2012). Se enrichment in soils is affected by many factors, such as soil parent materials, soil physical and chemical properties, farming pattern, and microbial activity (Wang Y H et al., 2022; Sharma et al., 2015). The utilization efficiency of Se by plants is determined mainly by the bioavailable Se forms rather than the total Se concentrations in soils, which means that the high concentration of bioavailable Se in soils is more conducive to the enrichment of Se in plants (Wang Q et al., 2022; Zhou et al., 2021; Dinh et al., 2019; Li et al., 2017).

Se biofortification is a promising strategy for Se enrichment in agricultural products. Traditional methods of agronomic Se bio-enhancement mainly include direct application of Se fertilizer into soils, the spray of Se fertilizer on foliage, and soaking of plant seeds in Se solutions(Li et al., 2021a, b). However, the direct inorganic Se fertilizer might not be an efficient and safe strategy due to its low utilization rate and uncontrolled amount of Se absorbed by plants (Muleya et al., 2021; Zhang et al., 2018; Wang et al., 2017). Application of inorganic Se fertilizers may also result in soil compaction and pH imbalance (Chen et al., 2021; Huang Y P et al., 2021; Zhang et al., 2020). Microorganisms play an essential role in improving the bioavailability of Se in the soil. The concentrations of organic matter, alkali hydrolyzed nitrogen, available phosphorus, and available potassium could be increased by adding microbial agents(Chen et al., 2021,Deng et al., 2021,Huang C L et al., 2021). However, at present, only a limited study focused on microbial agents for Se enrichment in crops. Microorganisms, including Se-oxidizing bacteria and Se-reducing bacteria, can play an essential role in the Se cycle in soils (Zhang J et al., 2019; Turanov et al., 2011). In anaerobic conditions, SeO₄^2-/SeO₃^2- can be used as terminal electron acceptors by microorganisms and reduced to elemental Se via dissimilatory reduction (Bao et al., 2014; Stolz et al., 2006; Hapuarachchi et al., 2004; Kashiwa et al., 2001; Huber et al., 2000; Ike et al., 2000). Some microorganisms can also reduce SeO₄^2-/SeO₃^2- to elemental Se under aerobic or microaerophilic conditions (Wadgaonkar et al., 2019; Song et al., 2017; Kuroda et al., 2011; Tejo Prakash et al., 2009; Hunter and Kuykendall, 2007). There are many pathways for the reduction of Se(IV), such as Painter-type reaction, thioredoxin system, dissimilatory reduction, siderophore-mediated reduction, and sulfate reduction (Wang D et al., 2022; Pettine et al., 2012; Hockin and Gadd, 2003; Lloyd, 2003; Stolz et al., 2002; Painter, 1941). Besides, microbial communities, including nitrogen-fixing bacteria and dissimilatory iron-reducing bacteria, can also enhance the availability of Se in soil (Wang Z et al., 2022). So far, many Se-reducing strains have been isolated from soil and water, such as Enterobacter cloacae SLD1a-1 (Losi and Frankenberger, 1997), Streptomyces sp. ES2-5 (Tan et al., 2016), Comamonas testosterone S44 (Zheng et al., 2014), Citrobacter sp. NDSe-5 (Won et al., 2021) and Pseudomonas sp. NDSe-2 (Won et al., 2021). These strains could reduce 40%-98% of 1.0 mM Se(IV). However, whether these functional strains can improve soil Se availability and their fortification of Se in crops are still not well understood.

The current study described a novel Se(IV)-reducing strain isolated from Se-enriched soil, which could produce Se nanoparticles (SeNPs) and further promote bioavailable forms of Se in soil and the Se concentrations in Brassica chinensis L. plants. Results of this study can help us better understand the microbially mediated Se transformation and might provide a prospective strategy for microbial fortification of Se in agricultural products.

1 MATERIALS METHODS

1.1 Soil Collection and Strain Isolation

Soil samples were collected from a Se-rich area located in Jinhua City of Zhejiang Province, China. Soils for strain isolation were collected from 10–20 cm of plant roots in dry land, which were mainly grown with vegetables. The total Se concentration in this soil was 0.46 mg/kg, while the bioavailable Se (the sum of soluble Se, exchangeable Se and carbonate-bound Se) was 0.11 mg/kg. To avoid contamination, soil samples for strain isolation were quickly placed into 50 mL sterile polypropylene tubes and stored in ice boxes (4 ^oC). Bulk soil was collected at the same site for pot experiments. All samples were transported to the lab within two days for further experiments.

Se(IV)-reducing bacterium was isolated on 1/10 TSA tryptic soy agar (TSA, pH 7.3, Difco) medium containing 1.0 mM Na₂SeO₃ (Tan et al., 2016). Weighed 10 g of collected soil and mixed with 90 mL sterile pure water, shook at 150 rpm for 5 min, and settled for 10 min. The supernatant was taken for gradient dilution and spread evenly onto a solid 1/10 TSA medium. After 48 h of incubation at 30 ^oC, visualized colonies with reddish color were selected and separated for several times until a single colony appeared on the solid medium. The single colony was cultured into log-phase in liquid 1/10 TSA medium at 30 ^oC for subsequent experiments.

1.2 Strain Identification

Physiological and biochemical identification of the strain was carried out according to previous studies (Huang C L et al., 2021; Jiang et al., 2015). Colony morphology was determined visually, and microscopic characteristics were observed under the optical microscope and compared with the “Bergey’s Manual of Systematic Bacteriology 9th Edition”. The detailed strain morphology was further observed with 100 kV TEM (Hitachi HT-7700, Japan). Microbiochemical identification tubes (Hangzhou Microbial Reagent Co., LTD, China) were used to identify the chemical and carbon metabolic characteristics of the strain compared with the characteristics of other isolates identified as the same genera.

The genomic DNA of the strain was extracted using miniBEST Bacteria Genomic DNA Extraction Kit (Takara, Japan) according to the manufacturer’s instructions. The DNA concentration and quality were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Universal primer set 27F (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ) and 1492R (5ˊ-GGTTACCTTGTTACGACTT-3ˊ) were used to amplify bacterial 16S rRNA genes (Weisburg et al., 1991). Detailed information was provided in the Supporting Information. The 16S rRNA gene sequence has been deposited in the GenBank database under accession number MW846086.

1.3 Se(IV) Reduction Assay

Se(IV) reduction of the strain was performed with chemical defined medium (CDM) modified with 1.0 mM Se(IV) under aerobic conditions. Cells in 5 mL cultures were centrifuged, washed with physiological saline, and inoculated into 50 mL CDM containing 1.0 mM Se(IV). A sterile medium was used as a control. All experiments were conducted in triplicate under 150 rpm at 30 ^oC. It was taking 1 mL of the mixed solution every 6 h to determine Se(IV) concentration and biomass variations. The sampled liquid suspensions were used to determine Se(IV) concentration after being filtered with 0.22 μm filters to exclude cells. The samples were centrifuged to obtain cell deposits and washed three times with physiological saline. The intracellular protein of bacterial cells was extracted by ultrasonic fragmentation for 3 min (300 w power, 3 s/7 s), and its biomass concentration was quantified with Pierce^® BCA Protein Assay Kit (Thermo Scientific). The Se concentrations were determined by hydride generation-atomic fluorescence spectrometry (HG-AFS, Hai Guang AFS-9780, Beijing, China).

Log-phase (24 h) cells of B.pseudomycoides SA14 were obtained by growth at 30 ^oC, shaking at 150 rpm in 100 mL CDM broth. The modified method was based on a previous study (Zheng et al., 2014). The cells in 100 mL CDM cultures were harvested by centrifugation for 10 min at 8 000 × g, the supernatant was removed. After being harvested, the cells were suspended in fresh sterile CDM broth. The suspension was placed on ice and treated with ultrasound for 20 min (20 amplitude micron, 5 s/5 s). This experiment set up four different systems: no cell control, live cells, heat-deactivated cells, and broken-cells suspension. Each design was added with 0.2 mM Se(IV) and incubated at room temperature for 12 h.

1.4 Identification of Reduction Products of the Strain

Strain B. pseudomycoides SA14 was incubated in CDM broth with 1.0 mM Se(IV) for 6 h. The bacterial suspension was centrifuged (8 000 × g, 4 ^oC, 10 min) and washed three times with phosphate buffer (PBS, pH = 7). The strain bullets were prepared for TEM observation (Hitachi HT-7700, Japan) and EDS analysis (Oxford X-Max, UK).

SeNPs produced by the strain were collected according to the method described by Dobias et al. (2011). Briefly, the cultured suspension was centrifuged at 4 ^oC, 8 000 × g for 10 min to remove biomass. The supernatant was then centrifuged at 4 ^oC, 20 000 × g for 15 min to collect SeNPs. The obtained SeNPs were observed with the scanning electron microscope (SEM) (Hitachi SU8010, Japan) and analyzed by spot scanning and area scanning with energy dispersive spectroscopy (EDS) (Hitachi SU8010 with accessory XPS, Japan).

1.5 Pot Experiments

Brassica chinensis L. was selected for pot experiments. Details about the cultivation and management of the plants were provided in the Supporting Information. Four experimental groups were designed to investigate the effects of the strain on Se enrichment in soils and plants, which included the P + B group (Brassica chinensis L. planted with SeRB added), P group (only Brassica chinensis L. planted without SeRB added), B group (only SeRB added without Brassica chinensis L. planted) and Control group (no Brassica chinensis L. planted and no SeRB added). Duplicates were set for each group. The cultivation conditions of these pots were: 25 ^oC for 14 h in light (240 μmol/m²/s) and 20 ^oC for 10 h in darkness, with 60%–70% of field capacity.

About 10 g of soils samples adjacent to the plant roots were taken on the 1st, 5th, 10th, 15th, and 30th day, respectively, after the SeRB was added. Samples were quickly stored in the refrigerator at -20 ^oC for subsequent Se form determination and microbial communities analysis. After 30 days of cultivation, the total Se concentrations of the above-ground parts of Brassica chinensis L. in the P group and P + B group were measured using AFS. Details about the extraction and determination of Se concentrations in soils and plants were provided in the Supporting Information.

To explore whether the SeRB added to the soil could survive and its influence on the original soil microbial communities, the soil microbial communities of the P group and P + B group on the 1st day and 30th day were analyzed. Soil genomic DNA was extracted according to the method mentioned above. The forward primer 341F (5-CCTACGGGNGGCWGCAG-3′) and reverse primer 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used to amplify the V3-V4 region of the microbial 16S rRNA genes. After purification, 25 ng of purified amplicon were sequenced on the illumine Miseq platform. The raw sequencing data have been uploaded to NCBI's Short Read Archive database with the accession number PRJNA730710.

2 RESULTS AND DISCUSSION

2.1 Bacterial Identification

A novel isolated Se(IV)-reducing strain was obtained and referred to as SA14. The 16S rRNA gene sequence of SA14 showed 99% sequence similarity with the other B. pseudomycoides strains from the GenBank database, such as B. pseudomycoides strain 74 (MH910178) and B. pseudomycoides strain JBRI-MO-0005 (MK302220). The neighbor-joining phylogenetic tree was constructed based on the 16S rRNA gene sequences of these closely related strains from the GenBank database (Figure 1). Phenotypic analysis showed that the strain was a gram-positive bacterium (Figure S1a). The strain was rod-shaped and 2.8 μm × 1.2 μm (Figure S1b). Its optimal growth conditions were temperature of 30 ^oC and pH of 7.0 (Figure S2). The primary biochemical and physiological characteristics of SA14 were not the same of isolates from the same genus (Table 1). Compared to B. oceani SW109T and B. oceani W006 (Song et al., 2016), strain SA14 was positive for hydrolysis of arginine, ornithine and lysine, utilization of urea and sucrose, and negative for activity of oxidase. Compared to B. halodurans LMG 7121T (Nielsen et al., 1995), strain SA14 was detected positive for hydrolysis of arginine, utilization of citrate and urea, and negative for utilization of maltose, activity of β-galactosidase and oxidase. Similarly, compared to B. okuhidensis DSM 13666T (Li et al., 2002), strain SA14 was positive for hydrolysis of arginine, utilization of urea, and negative for utilization of maltose, activity of β-galactosidase and oxidase. Consequently, strain SA14 was characterized as B. pseudomycoides based on the phylogenetic, morphologic and physiologic characteristics.

2.2 Se(IV) Reduction and Biogenesis of Elemental Se Nanoparticles

B. pseudomycoides SA14 could reduce more than 93.48% of 1.0 mM Se(IV) within 54 h (Figure 2a). The maximal reduction rate was 1.65 μM/(μg protein·h) (Figure 2b). The strain presented a distinct stronger Se(IV) reducing ability than B. cereus AJK3, Comamonas testosteroni S44, Comamonas testosteroni SE26, and Chitinophaga sp. SE06 were reported in previous studies (Huang C L et al., 2021; Zheng et al., 2014). As far as we know, this was the first report of Se(IV) reduction by B. pseudomycoides. Accompanied by Se reduction, the cultured suspension changed into red color, which indicated the possible production of SeNPs. We could observe SeNPs produced inside and outside of cells by TEM and EDS (Figure 3). The live cells and broken-cells suspension showed Se(IV)-reducing ability while no cell control and heat-deactivated cells did not (Figure S5). There are many vacuole-like sturctures in the cells, which may be related to the detoxification of Se(IV) (Figures S1a, 3a and 3b) (Yin et al., 2019). The red products produced by the B. pseudomycoides SA14 were further analyzed by SEM-EDS. The results showed a strong absorption peak in 1.37 keV, and weak absorption peaks in 11.22 and 12.49 keV, which belonged to the Se element (Figure S3 and S4). In the spot scanning, the Se element accounted for 37.26% (spot 1) and 42.24% (spot 2) of the total element concentrations, respectively (Figure S4). Flat scanning was further used to analyze the distribution of Se in cell products. Strong Se signals (in red, Figure S4c) were shown in the elemental maps. Carbon (C) (Figure S4d), oxygen (O) (Figure S4f) signals were also exhibited in the elemental maps because nano-Se produced by microbial reduction of Se(IV) was usually coated with a protein layer which functioned as the capping agent during the formation of particles (Zhang et al., 2012). These results showed that the reduction of Se(IV) by B. pseudomycoides SA14 was an enzymatic reaction, and SeNPs were synthesized by microorganisms in the cell and then released out of the cell.

2.3 Effects of the Strain Addition on Se Concentrations in Soils and Brassica chinensis L.

Pot experiments were carried out to investigate the effects of the SeRB on the concentrations and forms of Se in soils. The concentrations of seven different forms of Se were detected in the Brassica chinensis L. rhizosphere soils when the Brassica chinensis L. plants were incubated with/without the SeRB. Soluble Se indicated 26.72% and 23.13% increase in group B and P + B, respectively, compared to 3.76% decrease in group P (Figure 4a). Exchangeable Se decreased by 3.13% in group P, whereas it increased by 30.63% in group B and 22.05% in group P + B (Figure 4b). Carbonate-bound Se increased 10.18% in group B and 30.89% in group P + B, respectively (Figure 4c). The concentration of bioavailable Se (the sum of soluble Se, exchangeable Se and carbonate-bound Se) in group B and group P + B increased obviously (Figure 4d). In contrast, humic acid-bound Se, which accounted for the largest proportion of Se at the beginning, decreased by 4.82% and 14.39% in group B and P + B, respectively (Figure S6b). The concentrations of organic matter (OM)-bound and residual Se showed no apparent variations among the four groups, while Fe-Mn-bound Se showed a slight decrease (Figures S6a, S6c and S6d). No apparent variation of Se forms was found in controls. In acidic soils with strong reducibility, Se mainly exists in the form of Se (IV), which is easily absorbed by clay minerals, ferromanganese, hydroxides, humic acid, and organic matter (Kamei-Ishikawa et al., 2007). Therefore, the addition of Se(IV)-reducing strain might, on the one hand, break down the humic acid-bound Se in the soil and release more bioavailable Se (Kulikova and Perminova., 2021; Ryu et al., 2021; Wang et al., 2016), on the other hand, produce SeNPs which showed a higher bioavailability than that of Se(IV) in soils (Golubkina et al., 2012).

The effects of SeRB on Se uptake by Brassica chinensis L. plants and the condition of plants growth were also investigated (Figure 5). Inoculated plants showed significantly higher Se concentrations than uninoculated controls. The total Se concentrations of plants in group P + B with SeRB were 59.48 μg/kg, which was 2.46 times higher than the Se concentrations of plants in group P (Figure 5a). The fresh and dry weights of plants in group P + B were higher than that in group P, indicating better growth conditions in group P + B (Figure 5b). The amendment of SeRB showed a better promotion of plant Se concentration compared with a previous study. For example, using soil Se fertilizer and foliar Se fertilizer increased 15.7% and 28.1% of the total Se concentration in oat, adding Se fertilizer to the soil increased the total Se concentration of maize by 20 μg/kg (Li et al., 2021a; Chilimba et al., 2012). These results indicated that adding SeRB could effectively promote Se enrichment in plants, which might have great application potential.

2.4 Changes of Soil Microbial Communities in Soils

This study explored the changes of microbial communities by analyzing the composition of microbial communities in soils at the 1st and 30th day in group P and group P + B. Microbial taxonomic analysis showed that four soil samples contained 516 OTUs, which could be divided into 9 phyla (Figure 6a). In the four soil samples, the main dominant phyla included Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Firmicutes. Compared with 1st day, the relative abundance of dominant phyla, including Proteobacteria, Actinobacteria, and Firmicutes, varied greatly. The relative abundance of Firmicutes to which the added SeRB belonged increased from 3.23% to 5.55% in group P + B. In contrast, the relative abundance of each phylum in group P without SeRB varied a little, which might be because group P was thought to be less disturbed, so the microbial communities in the soil remained relatively stable.

The top 16 genera with relatively high abundances were selected to analyze the variation of soil microbial communities at the genus level (Figure 6b). There were differences in the microbial communities between group P1 and group P1 + B on 1st day. This may be because the samples collected on 1st day were taken 24 h after SeRB was added to group P + B, and the initial addition of SeRB had some effect on the soil microbial communities. Compared with group P1, the relative abundance of Bacillus, Comamonas, and Dyella in group P1 + B was significantly increased. After 30 days cultivation, the relative abundance of Bacillus, Comamonas and Dyella was further increased. The relative abundance of Bacillus increased from 0.97% to 2.08%. The relative abundance of Comamonas increased from 1.03% to 15.50%. The relative abundance of Dyella increased from 11.51% to 28.48%. The relative abundance of Bacillus, Comamonas, and Dyella in group P after cultivation showed no apparent variation from that before cultivation. The microbial communities structure of group P remained stable. SeRB belonged to Bacillus (Figure 1). Its addition increased the relative abundance of Bacillus in group P1 + B. Bacillus, Comamonas, and Dyella are common genera in Se-rich soil, and all possess the function of Se metabolism (Wang Y H et al., 2022; Ullah et al., 2020; Zheng et al., 2014). Comamonas and Dyella were mainly distributed in soils enriched with high concentrations of exchangeable Se and carbonate-bound Se (Wang Y H et al., 2022). The addition of B. pseudomycoides SA14 promoted the increase of exchangeable Se and carbonate-bound Se (Figures 4b and 4c), thus increasing the relative abundances of Comamonas and Dyella. Previous studies indicated that Bacillus, Comamonas, and Dyella could promote plant growth by secreting indole-3-acetic acid (IAA), gibberellin and dissolving phosphorus in the soil, which is consistent with the better growth of Brassica chinensis L. planted with SeRB in the present study (Figure 5b) (Domínguez-Castillo et al., 2021; Rolewicz et al., 2018; Erturk et al., 2010).

3 CONCLUSION

A new isolate of B. pseudomycoides from Se-rich soils was found with strong Se(IV)-reducing capacity. The strain could reduce more than 93.48% of 1.0 mM Se(IV) within 54 h and generate SeNPs. Amendment of the strain to soils could distinctly increase the bioavailable Se in soils and Se concentrations in Brassica chinensis L.. Microbial communities analysis showed that Bacillus, which the strain belonged to become the dominant communities in soils after the strain addition. This study expands the current understanding of microbially-mediated Se transformation and may provide a reliable strategy for microbial fortification of Se in agricultural products.

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Funding

the Open Project of Technology Innovation Center for Ecological Evaluation and Remediation of Agricultural Land in Plain Area, MNR(ZJGCJ202001)

Basic Public Welfare Research Program of Zhejiang Province(LGF22D030001)

Jiande City(HX2022B-011)

RIGHTS & PERMISSIONS

China University of Geosciences (Wuhan) and Springer-Verlag GmbH Germany, Part of Springer Nature