Remediation of copper-contaminated soils and growth enhancement of Pakchoi (B. chinensis L.) via biofertilizers composed of new Bacillus amyloliquefaciens SYNU1

Background Excessive copper contaminants are harmful to soil, microbes, plants and humans and can be remedi-ated by biosorption. Applying biofertilizers to remediate copper-contaminated soil is an environmentally friendly way. Results In this study, we identified a new strain, Bacillus amyloliquefaciens SYNU1, based on morphological, biochemical, physiological and phylogenetic analyses. It has been cultured on Luria–Bertani (LB) medium and absorbs soluble Cu 2+ from pH 3.5–5 and 25–40 °C at Cu 2+ concentrations of 100 mg L −1 . The results showed that the maximum adsorption capacity of copper by strain SYNU1 is 53.09% w/w. Furthermore, microbe fertilizers were made based on strain SYNU1, these fertilizers were allowed to ferment for 15 days, and they were used for remediation of copper-contaminated soil and growth tests of Pakchoi ( B . chinensis L) in pot experiments. The results showed that the growth of Pakchoi planted in copper-contaminated soil at concentrations ranging from 50 to 200 mg kg −1 was inhibited, and its growth indices, such as plant height, fresh weight and dry weight, decreased significantly with increasing copper concentration. Compared with the control, the increases in plant height, fresh weight and dry weight of Pakchoi treated with biofertilizer were 10.37, 65.3 and 67.78%, respectively, indicating that biofertilizer could significantly promote the growth of Pakchoi.


Introduction
Moderate amount of copper is an essential micronutrient for the growth and development of all biological organisms, and it exists in many oxidation states and plays key roles in numerous physiological processes, such as photosynthesis, respiration, hormone perception, cell wall metabolism and oxidative stress protection in plants (Yruela 2005;Pilon et al. 2006).At the cellular level, copper also plays a signalling role in transcription and protein trafficking machinery, oxidative phosphorylation and iron mobilization (Puig et al. 2007).Thus, plants need copper to maintain normal growth.When copper iron is not available, plants develop topical symptoms, such as apical meristem necrosis and young leaf wilting (Liu et al. 2013;Hansda et al. 2015;Leng et al. 2015;Choinska-Pulit et al. 2018).
However, high concentrations of copper have high toxicity in plants, which inhibits plant growth and impairs important physiological processes such as photosynthesis and respiration.High levels of copper are also highly toxic in humans and can accumulate in the brain, skin, liver, heart muscle and pancreas, causing great harm to human health (Patsikka et al. 2002;Yruela 2005;Pilon et al. 2006;Puig et al. 2007;Stern et al. 2007;Yruela 2009;Marques et al., 2019;Hossain et al. 2020;Gong et al. 2021).
During the development of agriculture, excessive use of Cu-based pesticides and fertilizers and wastewater irrigation have resulted in high copper concentrations in agricultural land, which have decreased the land quality, reduced the crop yield and caused great economic losses.Copper ions are enriched and transferred through the food chain, posing a great threat to environmental safety and human health.Therefore, it is very important and necessary to remediate copper-contaminated soil.Traditional methods require complicated equipment, are expensive and easily produce secondary pollution.In contrast, bioremediation has become a hotspot in the remediation of heavy metal pollution due to its advantages of environmental friendliness, low cost, wide source of materials, short remediation period and easy operation (Özdemir et al. 2013;Verma et al. 2013;Han et al. 2020;Yin et al. 2021;Li et al., 2022;Wang et al., 2022).
Applying biofertilizers to remediate heavy metal-contaminated soil is an environmentally friendly, cost-effective and sustainable option.Biofertilizers contain living microorganisms and organic matter, which can convert the available metal ions in soil into stable metal ions, reducing their biological availability.In addition, applying biofertilizers can improve soil nutrients and provide nutrients for plant growth.Remediating heavy metalcontaminated soil by using biofertilizer not only has the advantages of easy operation, environmental friendliness and no secondary pollution but can also promote the growth of plants and improve the disease resistance of plants (Gajdos et al. 2012;Bhardwaj et al. 2014;Derakhshan Nejad et al. 2018;Wang et al. 2019).Therefore, in this study, we hypothesized that biofertilizer could be effective in immobilizing soil Cu and alleviating its toxic effect on Brassica campestris ssp.chinensis L. This effect is related to biofertilizers reducing the copper content of the bioavailable state in soil, improving B. chinensis L antioxidant enzyme gene expression and improving antioxidant contents.
The purpose of this study was to evaluate the effectiveness of biofertilizers on the remediation of copper-contaminated soil.The antioxidant enzyme gene expression and antioxidant content in B. chinensis L. were evaluated.The information provided in this paper is useful for providing a theoretical basis and technical support for safe remediation strategies for soil heavy metal pollution.

Molecular identification of the SYNU1 strain
The SYNU1 strain was provided by the Key Biological Laboratory of Shenyang Normal University.Initial cultures were revitalized from glycerol stocks stored at − 80 °C by streaking onto LB agar plates and incubating at 37 °C for 24 h.The cultures were regularly transferred to fresh LB medium every 2 to 4 weeks to maintain viability and ensure purity.Before each experiment, a single colony was picked from the fresh LB agar plate to start a pre-culture.After incubation at 160 rpm and 37 °C for 18 h, the culture medium was centrifuged at 1200 rpm for 2 min using the high-speed refrigerated centrifuge (Eppendorf 5427R, Germany), and the SYNU1 cell precipitate was obtained.A bacterial genomic DNA extraction kit (OMEGA, China) was used to extract genomic DNA.Universal primers for the 16S rDNA gene (27f and 1492r) and gyrB gene (UP1 and UP2r) were used for amplification of genomic DNA by polymerase chain reaction (PCR) (Singh et al. 2013).PCR was carried out by Bio-Rad CFX96 PCR system (USA) with 10 μL Premix Taq, 0.5 μL 27f and 0.5 μL 1492r, 8 μL ddH 2 O and 1 μL DNA as template.
The PCR procedure of the 16S rDNA gene was conducted as follows: 95 °C for 5 min, then 30 cycles of 95 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min.The PCR procedure of the gyrB gene was conducted as follows: 95 °C for 5 min, then 25 cycles of 93 °C for 60 s, annealing at 50 °C for 60 s and extension at 72 °C for 70 s, and a final extension at 72 °C for 10 min.The PCR products were analysed by 0.8% (w/v) agarose gel electrophoresis.The target gene fragments were cut, purified and recovered using a PCR Cleanup Kit (OMEGA, China).The recovered products were connected to the pMD18-T vector with size 2692 bp (Takara Bio Inc., Japan) and transformed into E. coli DH5α competent cells.Then, the competent cells were cultured for 1 h, and 100 μL of culture was applied to an LB plate containing ampicillin and incubated at 37 °C for 12 ~ 24 h.Colonies were selected for colony PCR and electrophoresis analysis in 1% (w/v) agarose gels.The colonies showing positive electrophoresis bands were sequenced by Thermo Fisher Scientific (China) Co., Ltd.The nucleotide sequences of the 16S rDNA gene and gyrB gene of the SYNU1 strain were uploaded to the Ezbiocloud (https:// www.ezbio cloud.net/) and NCBI (https:// www.ncbi.nlm.nih.gov/) databases for comparison.Homologous sequences were obtained, and a phylogenetic tree was constructed by Mega11 software (Tamura et al. 2021).
The SYNU1 strain was stained by the Gram method and observed by optical microscopy under an oil-immersion lens.The SYNU1 strain was subjected to a starch hydrolysis test, methyl red test, v-p test, indole test, sugar fermentation test, urease test, nitrate reduction test and citrate utilization test.

Biomass preparation
The SYNU1 strain was grown in an LB medium (LB medium: sucrose 2.0 g L −1 , yeast 4.0 g L −1 , peptone 6.0 g L −1 , MgSO 4 5.0 g L −1 ).The aseptic operation was carried out on a super-clean work table, and 100 mL of LB liquid medium was filled into a 250-mL triangular flask.The SYNU1 bacterial solution was inoculated at an inoculation rate of 1% (V:V).After incubation at 160 rpm by AEOLUS ™ IS-18CA/IS-18A Stackable CO 2 Incubator Shaker (USA) and 37 °C for 24 h, the cultures were centrifuged at 10,000 rpm and 4 °C for 15 min and washed triply with distilled deionized water to wash off adhering debris and culture medium.For determination of the cell dry weight of bacteria, the bacteria were oven-dried at 80 °C until a constant weight was attained (Li et al. 2017).
Copper stock solution 1.965 g CuSO 4 .5H 2 O solid was dissolved in 50 mL sterile deionized water at a concentration of 10 g L −1 , sterilized by filtration and stored at 4 °C.Other copper concentrations were obtained by diluting the stock solution with sterile deionized water.

Preparation of biofertilizers and copper-contaminated soils
The SYNU1 strain was grown in LB liquid medium at 180 rpm and 37℃ for 48 h.Chicken manure, rapeseed meal, rice bran, soybean meal, fish bone meal, peat charcoal and seaweed powder were mixed evenly in a proportion of 50:10:10:10:10:5:5, and 60% tap water and 5% (v/w) fermentation broth of SYNU1 strain were added.Then, the mixture was fermented at room temperature for 15 days, and the biofertilizer was obtained.
The soil samples (0 ~ 20 cm) used for laboratory experiments were collected from the test site of the Shenyang Normal University (41° 54′ 33″ N, 123° 25′ 37″ E), Liaoning Province, China.Soil samples were homogenized, air-dried and sieved to 2 mm.Copper-contaminated soils were prepared with increasing concentrations of copper ranging from 0, 50, 100, 150 and 200 mg kg −1 by using CuSO 4 .5H 2 O.The metal salt was dissolved in distilled water, sprayed on the soil sample and mixed thoroughly (Haneef et al. 2014).Then, copper-treated soil samples were incubated in a pot and kept for 30 days.Before planting, the actual copper concentrations in the incubated soils were determined using atomic absorption spectroscopy (AAS).

Pot experiment of B. chinensis L
Pot experiments were conducted with the incubated soil samples to evaluate the effect of biofertilizers on the remediation of copper-contaminated soil and the growth of B. chinensis L. Each pot was filled with 300 g of soil thoroughly mixed with 3% (w/w) biofertilizers.For each treatment, three replicates were kept.Control pots were kept for both treatments without copper.Fifteen-day-old healthy seedlings of B. chinensis L. was transplanted to the experimental pots.Plants were grown under natural environmental conditions for 30 days.The growth indicators of plants were recorded in each pot.

Soil samples and plant tissue analysis for copper
Representative samples of initial soil and biofertilizer were collected, air-dried and ground to 1 mm before chemical analysis.The pH (solid: deionized water = 1:5 w/v) of the soil samples was measured.The control soil and contaminated soil were analysed for copper concentration by atomic absorption spectroscopy.
The plants were harvested after 40 days of growth, and their growth indices and heavy metal contents in different parts were measured.The stem length was recorded, and the aboveground parts of the plants were washed very carefully with deionized water four times.Plant tissues were oven-dried at 80 °C, and the constant dry weights were recorded.The dried plant materials were ground to less than 1 mm for copper analysis.The plants were analysed for copper concentration by atomic absorption spectroscopy (UV-3600i Plus Spectrophotometer, Japan).

Gene expression analysis by real-time PCR
The leaves of B. chinensis L. were harvested after 40 days of growth, and leaves with treatment and without treatment were also collected for expression pattern detection.All the materials were first pulverized with liquid nitrogen and stored in a − 80 °C refrigerator.Then, total RNA was extracted from leaves by using the RNAprep pure plant plus Kit (Tiangen Biotech Co., Ltd).The concentration and quality of RNA were determined by a spectrophotometer and detected by agarose gel electrophoresis (Wang 2014).First-strand cDNA was synthesized from 2 mg of total RNA with MMLV reverse transcriptase and random hexamer primers (Takara) according to the manufacturer's instructions.The cDNA samples were used for real-time PCR with primers designed for the selected genes, and BcActin was used as a housekeeping gene (Table 1).The PCR was performed in a 20 mL reaction mixture containing 200 nM of each primer, 1 × SYBR Green PCR Master Mix (Takara, Dalian, China), and approximately 30 ng cDNA.Real-time RT-PCR was performed using the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA).The reactions were carried out as follows: 3 min at 95 °C for denaturation, 10 s at 94 °C, 20 s at 60 °C and 30 s at 72 °C for amplification for 45 cycles.The relative abundance of transcripts was calculated according to the software instructions in Bio-Rad CFX96 Manager.The specificity of each primer pair was verified by determining the melting curve at the end of each run and sequencing the amplified bands from gel electrophoresis.

Identification of isolate SYNU1 based on phylogenetic, morphological and biochemical characterization
The strain SYNU1 is a rod-shaped bacterium observed under an oil-immersion lens (Fig. 1A).A biochemical test was further employed to analyse the characteristics of this bacterium.The results clearly showed that the bacteria are an aerobic and gram-positive strain.The catalase test, citrate solution test, nitrate reduction test, V-P test, hydrolysis of gelatine test and starch hydrolysis test were positive, whereas the urease test and indole test were negative (Table 2).The bacterium can ferment the sugars glucose, maltose, starch and sucrose to produce acid.The 16S rRNA sequence of the SYNU1 strain was aligned to those available in the NCBI database.The alignment results showed that the isolated strain SYNU1 was closely related to Bacillus subtilis, Bacillus siamensis and Bacillus amyloliquefaciens, with an identity greater than 99%.A specific species cannot  Then, we spliced together the sequences of the 16S rRNA and gyrB genes of the SYNU1 strain and constructed a phylogenetic tree.The results showed that the SYNU1 strain and Bacillus amyloliquefaciens were clustered in the same group (Fig. 1B).

Effect of biosorption factors on Cu. 2+ biosorption by strain SYNU1
The effect of pH on the biosorption capacity of Cu(II) by strain SYNU1 is shown in Fig. 2A.It showed that upon increasing the pH from 3 to 5, the biosorption capacity increased first and then stabilized.The effect of different temperatures on Cu 2+ biosorption by strain SYNU1 is shown in Fig. 2B, revealing that the temperature had no significant influence Cu 2+ uptake.The adsorption capacity of copper by the SYNU1 strain at various initial Cu 2+ concentrations shows that with an increase in the initial Cu 2+ concentration from 40 to 200 mg L −1 , the biosorption efficiency decreases.A lower initial metal concentration resulted in a high adsorption rate.With increasing adsorbent biomass, the adsorption rate tends to rise first and then fall.The adsorption at different adsorbent dosages shows that the biosorption capability of Cu 2+ by strain SYNU1 increases with increasing adsorbent dosage.The adsorption capacity of copper by strain SYNU1 was 53.09% when the adsorbent biomass was 2.0 g L −1 .Fig. 2 The adsorption rate of copper by the SYNU1 strain under different adsorption conditions

Biofertilizer preparation
During biofertilizer fermentation, microbes decomposed the organic matter and released heat.In this study, the biofertilizer temperature increased rapidly during composting, peaked on the 5th day, and then decreased gradually with the maturation of biofertilizer (Fig. 3A).
The colour of the biofertilizer was initially greyish brown.
Because the humus content rose gradually during composting, the colour of the biofertilizer became dark brown (Fig. 3B).The odour of biofertilizers during preparation was pungent, which smelled strongly of ammonia.

Effect of biofertilizer on growth of B. chinensis L under copper stress
In this study, B. chinensis L. inoculated and not inoculated with biofertilizer showed significant differences in phenotypic traits under different copper concentrations (Fig. 4).The growth parameters, such as plant height, plant fresh and dry weight were significantly different among inoculated and non-inoculated biofertilizer at every level of copper treatment (Fig. 5), indicating that high copper concentrations could inhibit crop growth and decrease crop yield.When B. chinensis L was treated with different copper concentrations, the biomass of the treatment groups with biofertilizer was significantly higher than that without biofertilizer, indicating that biofertilizer can not only promote the growth of B. chinensis L but also alleviate the stress effect on B. chinensis L. We treated copper-contaminated soil with microbial fertilizer for 30 days, and the extractable Cu 2+ content of all soil samples was extracted with 0.1 mol L −1 HCl.The results showed that there was no significant difference in the content of soil extractable Cu 2+ between the treatments with biofertilizer and without biofertilizer when the copper concentration exceeded 100 mg kg −1 .

Expression level of antioxidant enzyme gene in B. chinensis L
Real-time PCR analysis showed that the expression of CuZnSOD increased with increasing copper concentration (Fig. 6).The expression of the CuZnSOD gene in B. chinensis L treated with biofertilizer was significantly lower than that without biofertilizer, which indicated that biofertilizer could reduce the toxicity of copper and alleviate copper stress on B. chinensis L. The expression of the BcCAT1 gene in B. chinensis L. increased when treated with biofertilizer but decreased when treated without biofertilizer with increasing copper concentration.The expression patterns of the BcAPX gene and BcGR gene in B. chinensis L. treated with biofertilizer Fig. 3 Changes in fermentation temperature and biofertilizer colour and without biofertilizer were similar (Fig. 6), and their expression levels decreased with increasing copper concentration.The leaves of B. chinensis L. used in this study were collected after 30 days of growth in coppercontaminated soil, which is different from previous studies.Under long-term stress, plants do not have enough energy to synthesize multiple antioxidant enzymes at the same time, and only one of the more efficient types can be synthesized to continuously remove ROS.

Discussion
Microbe is useful for removing heavy metals and organic contaminants from soil and water (Tang et al. 2021).16S rRNA gene sequencing is a widely practised technique for bacterial identification, but it has limitations for members of closely related species (Fox et al. 1992;Yamamoto and Harayama 1995).Some protein-coding genes, such as rpoB (Kim et al. 1999), gyrB (Yamamoto and Harayama 1995) and gyrA (Chun and Bae 2000), have been concluded to be useful for the identification of closely related taxa.The gyrB gene was used to identify the Bacillus subtilis group, which comprises eight closely related species with an identity range of 98.1-99.8% in 16S rRNA gene sequences, and the results showed that the range of gyrB gene sequence similarities among the eight type strains was 75.4-95.0%(Wang et al. 2007).This result indicated that the gyrB gene sequences provide higher resolution than 16S rRNA gene sequences (Fig. 1B).
The adsorption mechanism of heavy metals by microorganisms is a complex biological process that is affected by pH, temperature, adsorption time, metal concentration and adsorbent biomass.The analysis of those factors is important for evaluating the biosorption potential of biomaterials.This result was because higher concentrations of ionic copper was toxic to the bacterium and inhibited the normal growth and metabolism of the Bacillus amyloliquefaciens SYNU1, this was similar to previous studies (Peng et al. 2019).Furthermore, the transformation of Cu into a more stable form by microorganisms cannot be ignored (Sun et al. 2023).In this study, the temperature pattern was similar as that in previous studies (Chang and Yang 2009;Tsai et al. 2007) (Fig. 2).Among biocontrol agents, B. amyloliquefaciens is considered safe and to have high potential because it is ubiquitous in nature and exhibits high thermal tolerance by forming resistant spores.Generally, the main targets of Bacillus species biocontrol agents are powdery mildew and grey mould diseases (Tanaka et al. 2017).Because B. amyloliquefaciens has a strong inhibitory effect on Botrytis cinerea and Podosphaera fusca, B. amyloliquefaciens also has a significant effect on promoting plant growth and is often used to promote the growth of many crops, such as tomato, rice, cucumber and other crops.pH is an important factor that affects the solution chemistry of metals and the activity of the functional groups on the surface of microbial cells.It was established that a pH range between 3 and 6 was conducive to biosorption (Gadd and Rome 1988).Studies at pH values higher than 5 were not conducted because insoluble copper hydroxides precipitated and restricted true biosorption studies.It was established that temperature influences the biosorption process to a lesser extent within the range of 20-35℃ (Veglio and Beolchini 1997).The microorganisms and organic matter contained in biofertilizers have a strong adsorption effect on metal ions, which can reduce the availability of heavy metals in soil and reduce the number of heavy metals absorbed by crops.Therefore, the use of microbial fertilizer is an effective measure for the passivation and remediation of heavy metal-contaminated soil.It is reported that maize and sunflower were treated with 10 mg L −1 Cd 2+ , and compared with the control group, the application of biofertilizer could significantly reduce the absorption of Cd 2+ by corn and sunflower (Gajdos et al. 2012).Adding biofertilizer decreased the content of soil extractable Cd 2+ (Wang et al. 2019).We have also obtained similar results.Furthermore, when the copper concentration exceeds 100 mg kg −1 , it is inconsistent with previous research findings (Abbaszadeh-Dahaji et al. 2021).The likely reason for this is the use of different extractants.The strong acid HCl, which was used in this study, had a detrimental effect on soil pH, thereby facilitating the extraction of some Cu 2+ ions that were fixed by high pH.Another possibility is that the high concentration of Cu 2+ ions inhibits the normal growth and metabolism of Bacillus amyloliquefaciens SYNU1.
Copper (Cu) is an important essential micronutrient for plant growth and development.However, it can easily lead to poisoning of plants at high concentrations.A common consequence of Cu poisoning is the rapid and excessive accumulation of reactive oxygen species (ROS) due to interference with photosynthetic and respiratory electron transport activities, especially that of chloroplast membranes.High levels of ROS expose cells to oxidative stress, leading to cell membrane dismantling.It has been previously reported that plants resort to a complex defence mechanism to counteract oxidative stress caused by Cu exposure.For example, plants possess very efficient enzymatic antioxidant defence systems.The components of enzymatic antioxidants include SOD, CAT, APX, MDHAR, DHAR and GR, which work in concert to control the cascades of uncontrolled oxidation and protect plant cells from oxidative damage by scavenging ROS (Gill and Tuteja 2010).− by catalysing its dismutation, with one O 2 − being reduced to H 2 O 2 and another oxidized to O 2 (Bowler et al. 1994) (Willekens et al. 1997;Gill and Tuteja 2010).Ascorbate peroxidase (APX) is thought to play the most essential role in scavenging ROS and protecting cells in higher plants, which is involved in scavenging H 2 O 2 in the water-water and ASH-GSH cycles and utilizes ASH as the electron donor (Asada 1992).Glutathione reductase (GR) is a flavoprotein oxidoreductase found in both prokaryotes and eukaryotes.It is a potential enzyme of the ASH-GSH cycle and plays an essential role in the defence system against ROS by sustaining the reduced status (GSH) (Romero-Puertas et al. 2006).

Conclusion
In summary, the effectiveness of biofertilizer with SYNU1 on the growth of Pakchoi planted in coppercontaminated soil was evaluated.Based on morphological, biochemical, physiological and phylogenetic analyses, SYNU1 was identified as Bacillus amyloliquefaciens.SYNU1 has a high adsorption capacity for copper, the maximum adsorption capacity of copper by strain SYNU1 is 53.09% w/w.The inoculation with the biofertilizer prepared by SYNU1 significantly alleviated the toxicity of copper on B. chinensis L. by decreasing CuZnSOD gene expression, and increased plant height, plant fresh and dry weight which were attributed to the increasing of BcCAT1 gene expression.However, the available copper content of soil treated with biofertilizer was not significantly reduced compared with that of soil treated without biofertilizer.The reason for this is unclear and needs to be further studied.

BcAPX
Fig. 1 SYNU1 strain morphology and phylogenetic tree based on the sequences of the gyrB gene sequences

Fig. 4
Fig. 4 Effect of biofertilizer on the growth of B. chinensis L. under different copper concentrations

Fig. 6
Fig. 6 Effect of applying microbial fertilizer on antioxidant enzyme gene expression levels in B. chinensis L

Table 1
Primers for PCR of antioxidant enzymes genes in B. chinensis L

Table 2
Some physiological and biochemical characteristics of SYNU1 strain . Catalases (CAT) are indispensable for ROS detoxification during stress conditions.It can directly dismutate H 2 O 2 to H 2 O and O 2 .It has a high turnover rate, and one molecule of CAT can convert approximately 6 million molecules of H 2 O 2 to H 2 O and O 2 per minute