1. Introduction
Groundwater is a valuable natural resource with significant economic and social importance as it plays a crucial role in drinking water supply and global agricultural irrigation [1–2]. However, in recent decades, water resources have faced significant threats owing to unsustainable extraction and increased pollution levels. Factors, such as climate change, land-use change, and population growth, contribute to various challenges that affect both the quantity and quality of groundwater [3–4]. Among these threats, agricultural activities significantly contribute to groundwater pollution, primarily through the application of fertilizers and the spread of animal manure. Consequently, nitrate (NO3−) pollution has emerged as a major concern, with elevated nitrate concentrations becoming a common problem in many regions worldwide [5–7].
As groundwater pollution has become a severe global problem, the significant effects of high nitrogen fertilizer application rates on groundwater pollution have been investigated [8]. The excessive use of nitrogen fertilizers can cause severe damage to farmland, leading to pollution, and high nitrate levels in groundwater pose both public health risks and environmental hazards [9]. Acute toxicity can occur when individuals consume water with high nitrate concentrations, potentially leading to methemoglobinemia or “blue baby syndrome,” where blood cannot adequately transport oxygen to the body’s cells. Long-term exposure to elevated nitrate levels in drinking water has also been linked to an increased risk of various types of cancers, including gastric, colorectal, bladder, urothelial, and brain tumors [7]. Therefore, the concentration of nitrate in drinking water are regulated to minimize public health risks, with the maximum contaminant level at 10 mg/L (NO3-N) in South Korea [10–11]. In South Korea, it has been reported that the concentration of NO3− in groundwater was in the range of 0.1−325.1 mg/L in 3,928 groundwater monitoring wells, indicating that 25% of samples was over the maximum contaminant level of drinking water [11].
Among the various treatment methods for nitrate-contaminated groundwater, biological methods, such as aerobic denitrification, are known to be effective for the treatment of nitrogen contamination owing to their simplicity and ease of use, high energy efficiency, eco-friendliness, and low operation cost [12–13]. A lab-scale continuous experiment was conducted using Bacillus species, a group of typical gram-positive probiotics easily found in a variety of natural environments, to indicate the application potential of Bacillus subtilis (B. subtilis) JD-014 for the effective removal of nitrogen, and the removal efficiency of nitrate nitrogen (NO3-N) was 98.91% [14]. Furthermore, four potential candidate genes of periplasmic nitrate reductase (napA0, napA1, napA2, and napA3) were recently identified for aerobic denitrification [14].
Bishnoi found that soil harbors a vast array of microorganisms that play crucial roles in food and fiber production. Additionally, microorganisms are responsible for maintaining the global nutrient balance and supporting ecosystem functions [15]. Within the soil-plant system, plant growth-promoting rhizobacteria (PGPR), a diverse group of soil bacteria, engage in complex interactions in the rhizosphere, thereby influencing plant growth and yield [16]. In recent years, PGPR have emerged as a significant and promising tool for sustainable agriculture. PGPR play a crucial role in sustainable agriculture by releasing plant growth regulators, phytohormones, and other biologically active substances. They also manipulate endogenous phytohormone levels, enhance nutrient availability and uptake through fixation and mobilization, and mitigate the harmful effects of plant pathogens [17]. These diverse functions make PGPR a valuable tool for promoting plant growth, improving crop yield, and reducing the need for chemical fertilizers and pesticides, thereby contributing to environmentally friendly and economically viable agricultural practices.
Notably, Zaidi et al. reported that B. subtilis plays a significant role in facilitating nickel accumulation in mustard plants, contributing to the promotion of plant growth and increasing the capacity for bioaccumulation [18]. Additionally, the use of probiotic bacteria, particularly B. subtilis, can suppress pathogenic growth, improve nutrient assimilation, and enhance environmental conditions in aquaculture settings [19]. Therefore, investigating the application potential of B. subtilis in denitrification processes and the subsequent use of treated nitrate-contaminated groundwater for enhanced plant growth is essential.
In this study, we investigated the growth rate of B. subtilis in nitrate-contaminated groundwater (NO3−-GW) and the potential use of biologically treated nitrate-contaminated groundwater for enhancing plant growth. Real nitrate-contaminated groundwater was used to conduct all the bacterial growth experiments, bioremediation of the contaminated groundwater, and germination and plant growth tests. The objectives of this study were to: i) investigate the bacterial growth of B. subtilis with and without nitrate; ii) evaluate the effect of ions in groundwater on the bacterial growth rate; iii) monitor the uptake of nitrate and the concentration effect of nitrate during bacterial growth; and iv) apply the treated groundwater to the growth of kidney beans in soil.
2. Materials and Methods
2.1. Chemicals and Materials
Sodium nitrate (NaNO3, ≥98%), sodium sulfate anhydrous (Na2SO4, ≥99%), and magnesium chloride hexahydrate (MgCl2 6H2O, ≥99%) were purchased from Samchun Chemicals, South Korea. Sodium hydroxide (NaOH, ≥97%) and hydrochloric acid (HCl, 35%) were purchased from Daejung Chemicals & Metals, South Korea. Sodium chloride (NaCl, 99.5%) and calcium chloride (CaCl2, ≥95%) were purchased from Showa and Junsei, Japan respectively. D-(+)- glucose (≥ 99.5%) was purchased from Sigma-Aldrich, United States of America (USA). All solutions were prepared using deionized water (DIW; 18.2 MΩ), which was purified by an ultrapure filtration system (HUMAN POWER I+ Water purification, Korea). Nitrate-contaminated groundwater was sampled from a well in Hongseong City, South Korea (36° 35′ 54.3″ N, 126° 40′ 3.4″ E) (Fig. S1). The livestock industry and agricultural activity near this area have significantly polluted the local groundwater resources. The bacterial strain Bacillus subtilis (B. subtilis, KACC 13751, source: plant growth promotion) used in this study was obtained from the Korean Agricultural Culture Collection (KACC) in freeze-dried ampoule form. Tryptic soy broth (TSB; Soybean-Casein Digest Medium) was used as the medium for the pure-culture growth of B. subtilis.
2.2. Bacterial Culturing and Batch Tests
2.2.1. Pure culture of B. subtilis
For a pure culture of B. subtilis, we first prepared the culture medium by dissolving 3 g of TSB powder in 100 mL of DIW. After autoclaving and cooling of the medium, 1 mL of the bacterial suspension was inoculated into the sterilized TSB medium and incubated for 24 h at 37 °C under 100 rpm. The optical density at 600 nm (OD600) was measured during the growth phase using an ultraviolet (UV)–vis spectrophotometer (GENESYS 10S, Thermo) (Fig. S2(a)) [19].
2.2.2. Bacterial inoculation
Seven groups of sample media (NaCl, NaCl + MgCl2, NaCl + CaCl2, NaCl + Na2SO4, NaCl + NaNO3, NO3−-GW, and NaCl + NO3−-GW) were prepared to investigate the effect of ions on bacterial growth. Different ion concentrations were selected based on the ion content of the nitrate-contaminated groundwater (Table S1). Each sample (100 mL), containing a 0.1% filtered glucose solution, was autoclaved and cooled to room temperature. Subsequently, 1 mL of B. subtilis cultured in the TSB medium was obtained by centrifugation and inoculated into the prepared sample media. The prepared samples were incubated at 37 °C under mixing at 100 rpm. At the time of sampling, the bacterial suspension was collected and the OD600 values were directly measured to evaluate the bacterial growth. Subsequently, bacterial suspension was filtered using 0.2-μm polyvinylidene fluoride (PVDF) syringe filters (Whatman) to analyze the nitrate concentration of the prepared samples.
2.3. Experiment for Plant Growth by B. subtilis Treated NO3−-GW
To investigate the enhancement in plant growth by B. subtilis-treated NO3−-GW, we prepared a total of five samples of kidney bean (i.e., Phaseolus vulgaris L) planted in triplicated identical soils. Subsequently, the prepared samples were watered everyday by DIW, NO3−-GW, NO3− (90% DIW + 1000 ppm of 10% NO3− = 100 ppm NO3−), NO3− + B. subtilis, and NO3−-GW + B. subtilis. After the germination of the kidney beans, the size of each plant structure was measured. The germination rate was calculated by counting the number of germinated kidney bean seeds relative to the total number of kidney bean seeds initially planted in the soil. For each germinated kidney bean, the lengths of the root and stem and the size of the leaves (horizontal and vertical lengths of each point) were measured. Finally, the plant samples were dried in the dry oven for 24 h at 105 °C to measure the weight of biomass of each structure (Fig. S2(b)).
2.4. Analytical Methods
All the aqueous samples were filtered using 0.2-μm PVDF syringe filters (Whatman) before the analysis. The concentrations of the dissolved cations (Na+, K+, NH4+, Ca2+, and Mg2+) and anions (F−, Cl−, Br−, NO2−, NO3−, SO42−, and PO43−) were measured using ion chromatography (IC) (Metrohm, 883 Basic IC plus) with cation (Metrosep c4-150/4.0, Metrohm AG) and anion (Shodex IC Anion sep No. 82504A) columns. A mixture of nitric and dipicolinic acids was used as the cation IC eluent at a flow rate of 0.9 mL/min, and a mixture of Na2CO3 and NaHCO3 was used as the anion eluent at a flow rate of 0.7 mL/min.
3. Results and Discussion
3.1. Bacterial Growth in Nitrate-Contaminated Groundwater
As shown in Fig. 1, the measured OD600 values indicated the significantly different growth trend of B. subtilis in DIW and NO3−-GW. The sample of DIW showed almost no increase in cell number during the 100-h incubation period, whereas a rapid increase in OD600 value was observed in the sample of NO3−-GW after 40 h of incubation. These results indicated that the growth of B. subtilis can be significantly accelerated in NO3−-GW.
After analyzing the ion concentrations of the nitrate-contaminated groundwater (Table S1), the effects of individual ions on the growth of B. subtilis were investigated (Fig. 2). The effects of Na+ and Cl− were not considered in this experiment as these ions were already present in the growth media to avoid the inhibitory effect caused by cell lysis. Notably, the OD600 value in the absence of NaCl decreased compared with that in NaCl samples for all cations and anions tested in this study (Fig. 3). It has been reported that the number of cells (B. subtilis) increased with increasing NaCl up to 0.5 M [20]. Because we used the 0.17 M of NaCl (Fig. 3), the concentration we used might be in the range for the enhanced bacterial growth. As shown in Fig. 2(a), both Ca2+ and Mg2+ did not influence the growth of B. subtilis in the control experiment (Fig. 2(a)), indicating no significant effect of cations because they are essential nutrients for the bacterial growth [21]. In contrast, owing to the presence of NO3− in the growth medium, the OD600 value showed a significant increase after 40 h of incubation and reached 0.07 after 100 h (Fig. 2(b)). This dramatic increase may be because B. subtilis can utilize nitrate through the candidate genes of periplasmic nitrate reductase (napA0, napA1, napA2, and napA3) during aerobic denitrification [14]. Indeed, we observed the concentration of NO3− in NO3−-GW sample decreased to 84.31 mg/L in after the incubation, which was the clear evidence for aerobic denitrification.
3.2. Effect of nitrate concentration on the bacterial growth
Previous results have confirmed that the presence of nitrate in groundwater can significantly enhance the growth of B. subtilis. To investigate the effect of nitrate concentration on bacterial growth, different concentrations of nitrate (10 and 100 mg/L) were added to the DIW-based samples (Fig. 4). The nitrate sample with a concentration of 100 mg/L showed a very similar OD600 value (approximately 0.0275) to the NO3−-GW sample owing to the similar concentrations of nitrate in the real nitrate-contaminated groundwater (i.e., 103 ppm, as presented in Table S1). Notably, we observed a higher OD600 value (approximately 0.0360) in the nitrate sample with a concentration of 10 mg/L, indicating that high nitrate concentrations may inhibit bacterial activity. Notably, high nitrate concentrations are known to cause nitrite accumulation owing to the inability of bacteria to reduce nitrite as it is toxic [22]. The results revealed that a certain amount of nitrate could be effectively utilized by B. subtilis, which could be further applied to the denitrification process and enhance plant growth.
3.3. Enhancement in Plant Growth by Watering with Biologically Treated NO3−-GW
To examine the rate of plant growth, kidney beans were planted in triplicate and the germination process was observed daily. The samples of DIW and NO3−-GW + B. subtilis showed the earliest germination at day 3 (Fig. 5(a)). Furthermore, the visual plant growth after 7 days of planting was the best for all three samples of NO3−-GW + B. subtilis, which was comparable with those of the samples of DIW, wherein only one sample showed enhanced growth. These results indicate that B. subtilis-treated real NO3−-GW could be used to enhance plant growth without the problem of phytotoxicity. In contrast, the sample grown in NO3−-GW failed to germinate until day 7 (the second image of Fig. 5(b)). The germination and growth of kidney beans are known to be affected by various biotic and abiotic environmental stresses, including salinity, cyanobacteria, pathogenic fungi, and heavy metals [23–26]. Furthermore, we observed the germination of kidney beans in both of NO3− and NO3− + B. subtilis samples, which contained a similar concentration of NO3− ions (~100 mg/L). As mentioned earlier, the NO3− concentration in the NO3−-GW + B. subtilis after the incubation was measured at 84.31 mg/L, indicating the presence of remained NO3− in groundwater. This suggests that the inhibition of germination in the NO3−-GW sample may not only by the high NO3− concentration but also by other environmental stresses in the contaminated GW. It should be noted that B. subtilis was found to promote the germination and growth of kidney beans under both NO3− and NO3−-GW conditions. While the specific mechanisms of physiological actions are not fully understood in this study, B. subtilis has been known to produce a wide range of biologically active substances for antibiotic and phytohormone-like activities, capabilities of atmospheric N2 fixation, phosphate solubilization, and induction of systemic resistance/tolerance in plants [25,27]. This is why B. subtilis is called as PGPR influencing plant growth and yield. Thus, we suspect that the positive effects of B. subtilis may influence the activation of seed germination and plant growth in this study.
The sample with NO3−-GW + B. subtilis showed the longest lengths of roots and stems (Figs. 6(a) and (b)). The size of two leaves determined by two factors, namely, the vertical and horizontal lengths, indicated that the samples with NO3−-GW + B. subtilis showed the highest development of the structures of kidney beans (Fig. 6(c–f)). Fig. 7 showed that the dry weight of biomass for NO3−-GW + B. subtilis was considerably higher than those of other samples, not only for the total biomass (approximately 1.7−2.0-fold) but also for each plant structure (i.e., roots, stems, and leaves). The results obtained from this study show that real nitrate-contaminated groundwater can be treated with PGPR and PGPR-treated groundwater can subsequently be used for watering plants. We also concluded that the presence of other nutrients for plant growth in real groundwater could be the reason for the best plant growth in the sample with NO3−-GW + B. subtilis compared with that of the sample containing NO3− + B. subtilis.
4. Conclusions
Herein, we report the potential of a novel biological treatment method for the aerobic denitrification of nitrate-contaminated real groundwater and the secondary application of treated groundwater to enhance plant growth. The experimental results revealed that the growth rate of B. subtilis could be enhanced by the presence of nitrate, which was comparable with that of the case wherein cations showed no significant effects on plant growth. Furthermore, plant growth experiments using kidney beans showed that watering with B. subtilis-treated groundwater could be used to enhance plant growth, whereas germination failure was observed by direct watering with nitrate-contaminated groundwater without microbial treatment. This indicated that B. subtilis-treated groundwater can shorten the germination time of kidney beans and increase the total growth of plants, including the lengths of roots, stems, and leaves.
The experimental results of this study can be applied to the scenarios shown in Fig. 8: i) nitrate pollutants originating from livestock operations, excessive use of nitrogen fertilizers, and industrial wastes; ii) pumping of nitrate-contaminated groundwater and transfer to PGPR-containing basins or reactors; iii) aerobic denitrification; and iv) use of treated groundwater as agricultural irrigation water.
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