Potential Role of Combined Microbial Inoculants and Plant of Limnocharis flava on Eliminating Cadmium from Artificial Contaminated Soil

Abstract

This study aimed to investigate the possibility of eliminating cadmium (Cd) from contaminated agricultural soil using a combination of microbial inoculants of Penicillium chrysogenum fungus and Bacillus licheniformis with Limnocharis flava plants. Limnocharis flava (L. flava) and microbial inoculants with four Cd levels, including 2.05 (T1 control), 5 (T2), 10 (T3), and 20 mg/kg (T4), respectively, were tested in green house conditions. Cd accumulation was evaluated to assess the safety limit of the Cd concentration in the edible parts of L. flava. The results showed that the application of the microbial inoculants facilitated the stress tolerance of the plants caused by a Cd accumulation in the soil matrix. The use of Penicillium chrysogenum and Bacillus licheniform in combination with L. flava increased the Cd accumulation in plant biomass. The total Cd after the experiment varied between 4.86 and 18.44 mg/kg in dry, clean soil, equivalent to reduction rates of 2.80, 4.40, and 7.80%, respectively. Meanwhile, the availability of Cd in soil was significantly reduced by 12.50, 13.04, 13.33, and 13.93%, respectively. Consequently, the microbial inoculants helped the plants to grow healthily, increased the yield, and reduced the total and available Cd content of contaminated agricultural land up to a concentration level of 5 mg/kg.

1. Introduction

Along with rapid economic development, the construction of concentrated industrial zones and medical facilities as well as the expansion of agricultural production areas produce huge quantities of wastes containing heavy metals and other hazardous substances [1]. Heavy metal compounds in untreated or poor waste sources are gradually accumulated in soil [2]. The problem will become acute, especially when agricultural lands are contaminated with heavy metals, which can pose a direct threat to human health [3]. Hence, environmental protection and food safety are serious matters of urgency for every country [2,3]. Many countries in the world are facing problems of toxic metal pollution in agricultural soil [1,3]. Cadmium (Cd) in soil causes food contamination, thereby damaging human health through the food chain [1,4]. It is a typical heavy metal that exhibits toxicity due to its continuous accumulation in the environment. The cumulative tendency of this element in biomass and the food chain poses a serious threat to plants, animals, and humans even at low concentrations [2]. Therefore, to maintain a safe food chain and a healthy agricultural ecosystem, the removal of Cd from agricultural soil is essential [2]. In practice, the treatment of soil contaminated with heavy metals, particularly on a large scale, is a very difficult, expensive, and technically demanding procedure [2,3,4]. Therefore, the use of microorganisms or plants to treat heavy metals is a relatively cheap, safe, and effective method [5,6]. In this context, the combination of microorganisms and plants are used to restore a polluted environment to its original state [5]. In particular, the combination of plants and microorganisms is not only a means of promoting Cd removal [6], but also a way of increasing the activity and diversity of microorganisms in soil, leading to a healthy ecosystem.

A literature review revealed that the combined application of bacteria and plants could effectively promote plant growth and control Cd in soils [7,8,9]. For instance, two indigenous fungal species, Mucor Circinelloides and Trichoderma Asperellum, were used with Arabidopsis Thaliana for promoting the phytoremediation of Cd and Pb in soil [7]. Li et al. [10] investigated the tolerance of Aspergillus aculeatus to Cd and their observations showed that the plants inoculated with A. aculeatus exhibited a higher relative growth rate and a normalized relative transpiration rate under Cd stress than non-inoculated plants, regardless of genotypes. Additionally, the injection of Ralstonia eutropha and Chryseo bacterium humi in Zea mays L. resulted in increased plant biomass in shoots, and accordingly a higher accumulation of Cd in the roots [11]. Micrococcus sp. TISTR2221 also promoted root and shoot lengths, dry biomass, and accumulated Cd in the shoots of Zea mays. L. plants [12]. Nguyen and Stéphane combined mycorrhizal arbuscular fungi and ferns for removing Pb from soil and indicated that Pb accumulated up to 834.63 mg/kg in the roots and 121.19 mg/kg in the stalk leaves of the fern [13]. It was also demonstrated that Enterobacter sp. (S2) can promote plant growth and enhance Cd accumulation in rice plants [14]. Furthermore, Bacillus licheniformis and Penicillium chrysogenum were applied with many different plant species in order to promote plant growth under stressed soil conditions [15].

However, these studies only focused on the individual effects of fungi, bacteria, and plants on controlling Cd accumulation in soils. To the best of our knowledge, there is not sufficient information on the combination of different microorganisms, fungi, and bacterial strains for accumulating, eliminating, and treating heavy metal pollution in soils. Some plant, fungus, and microorganism species also have the ability to remove heavy metals, but they cannot survive under salinization and drought conditions in the Mekong Delta, Vietnam. The salinization and drought in the dry season has reduced agricultural plant growth and soil microorganism activity due to osmotic stress, low moisture, and toxic ions [16,17]. Therefore, native plants and microorganisms adapting to extreme soil and climate conditions and/or tolerant to high levels of heavy metals in soil have been selected in order to treat heavy metals in soil. Since heavy metal contamination has also been prevalent in Vietnam because of the dominance of agricultural activities [18,19], the remediation of soils polluted with heavy metals is of great importance.

L. flava, a wild aquatic flowering plant with tuberous roots developing into soil, is widespread in the Mekong Delta. This species is known as a metal hyper-accumulator because of its high capacity to treat Cd in soil, water, and wastewater [20]. It is widely employed as a forage in Southern Vietnam as well. There have been limited in-depth studies about the this species’ capability for Cd accumulation. Considering all the issues discussed above, research on bacteria and fungi in combination with L. flava for the accumulation of Cd and on ensuring forage safety for people is needed. Hence, in the current research, the indigenous fungus MHCM15-VN1 (Penicillium chrysogenum) and the strain MHCM7-VK2 (Bacillus licheniformis) were added to soil to improve the growth of L. flava and remove the effect of Cd [15,21]. Therefore, the aim of the present research was to investigate the effects of combined microbial inoculants with L. flava plants on eliminating Cd from artificially contaminated agricultural soil. It was hypothesized that the combined microbial inoculants with L. flava plants might be effective at eliminating Cd from contaminated soil for safe crop production.

2. Materials and Methods

2.1. Materials

The seeds of L. flava were purchased from the Southern Seed Company (SSC) in Vietnam. First, they were potted using unpolluted mud soil and placed in a shady place for one month in the campus of Hanoi University of Natural Resources and Environment in Thanh Hoa, Vietnam (20°05′04.1′′ N 105°51′34.6′′ E). Next, the seeds grew up to approximately 28 cm with either four or five leaves. These seedlings were then used as preliminary experimental materials.

The microbial inoculants were tested in the laboratory of the Institute of Soil and Fertilizers in Ha Noi, Vietnam. Then, pH neutralization was performed using 1% sugarcane rust and rice bran as carriers, with the microbial inoculants consuming peat. They consisted of two microbial strains of fungi, including MHCM15-VN1 and bacterium MHCM7-VK2 with microorganism density >4 × 108 mg/L. These strains were taken either from in-root or out-root soil samples located in agricultural fields in Ho Chi Minh City, Vietnam. They were isolated on glucose yeast extract agar (GYA) medium, according to the method presented by Vijayaraghavan and Yeoung [22]. Their ability to accumulate Cd in microbial cell biomass in the laboratory was tested by the liquid method expressed by Malik and Jaiswal [17] at Cd concentrations of 2.05, 5, 10, and 20 mg/L, consistent with QCVN 03-MT: 2015/BTNMT.

Methods of polymerase chain reaction (PCR), genomic sequence, and BLASTN program were used to identify the respective two strains of microorganisms, the PCR products of 16S and 28S after being purified, and the sequence was analyzed by BigDye 3.1 on 3130xl (ABI, Foster City, CA, USA). We used the version 2.1.2 of BLAST program to detect sequences that were similar to the research sequences published in gene banks http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome (accessed on 13 August 2022) to confirm the target sequence and to select some highly homologous sequences for comparison and segmentation. Additionally, version 7.0 MEGA 7 (Los Angeles, CA, USA) genealogy and evolutionary analysis program was used to determine the genetic relationship between the two analyzed strains. MHCM15-VN1 strain had an almost 100% similar sequence to the fungus Penicillium chrysogenum, while MHCM7-VK2 strain showed a 99.73% similar sequence to the bacterium Bacillus licheniformis, which was used for microorganism culture. The mixture of soybean meal, molasses, and sodium was used as a nutrient source to maintain the growth of microorganisms. After filling the bag with the nutrient compounds, the two microorganism strains were injected directly into bags at the ratio of 1 (microorganisms): 4 (nutrient compounds). Then, the bags were closed, sealed, and incubated for one week at room temperature before use. The mixed biomass of two strains of microorganisms, after level 2 propagation, was injected directly into the treated carrier bag and disinfected at the ratio of one part microbial fluid mixed with four parts carrier. Later, the microbial inoculant was closed and incubated for one week at room temperature before use. Microbial inoculant containing 2 g/kg dry soil was applied, which was the recommended level after testing the effect of microbial inoculant levels based on plant growth and accumulation of Cd by L. flava plants.

2.2. Pot Experiments

These experiments were carried out in the net house of the Department of Environment, a branch of Hanoi University of Natural Resources and Environment in Thanh Hoa, Vietnam. The experiment was designed with a randomized complete block (RCB) by IRRISTAT 4.0 software. Sixteen Styrofoam pots were divided into four groups (T1–T4) with different levels of Cd. Each group contained four Styrofoam pots with similar Cd concentration and was individually sampled to evaluate the Cd concentration in soil and plants.

Each treatment was conducted in quadruplicate in Styrofoam pots of 45 × 30 × 30 cm³, containing 10 kg of dry mud soil contaminated with Cd. The soil samples were naturally dried, smashed, and then passed through a 2 mm sieve to remove stones and gravel. Next, they were sterilized by autoclave at 121 °C (1 atm) for 30 min before they were placed in the experimental pots. The cadmium acetate solution, Cd (CH₃COO)₂, was mixed with the soil. The Cd content was prepared using the Vietnamese standards QCVN 03-MT:2015/BTNMT, whereby the T1 group treatment retained its original heavy metal content with 2.05 mg/kg (M1) of the soil experiment.

In order to achieve the concentration values of 5, 10, and 20 mg/kg Cd, the concentration of Cd(CH3COO)₂ added to the soil was calculated using the following formula for the Cd content in the dry soil remaining in T2, T3 and T4:

M (mg/kg) = (M1 − M2) × 113/204

(1)

where M is the amount of Cd to be added to the soil, M1 is the desired exprimental concentration of Cd in soil (5, 10, 20 mg/kg Cd), and M2 is the concentration of available Cd in the natural soil (Cd content of T1).

The Cd(CH₃COO)₂ was mixed well in the soil to restore the soil bioactivity. The mixture was examined for two weeks at 30 °C in 60–70% humidity. Then, 2 g of the inoculant was mixed with 50 g of the manure to fertilize the soil in each pot. Three seedlings of L. flava were planted in each pot so that the plants clung firmly to the soil in order to develop their roots and grow healthily. Three days after planting, the plants in the pot were irrigated with 5–10 cm of clean tap water (from the ground to the water surface) during the experiment (Figure 1). Furthermore, the morphology of L. flava during harvest time is shown in Figure 2.

2.3. Analysis of Cd Content in L. flava Plants after Harvest

The samples were harvested 60 days after growing. The harvested samples were washed with tap water and then rinsed using distilled water. The root, stems, leaves, and flowers were weighed by technical balance model XB2200C (Shanghai Jingke Tianmei Trading Co. Ltd., Shanghai, China). The roots, stems, leaves, and flowers were shredded and labeled using silver paper bags and then dried at 75 °C using drying oven model UF110. The drying did not stop until the weight became constant. In a consecutive process, the dried samples were transferred to a desiccator for cooling before recording the dry mass, then grounded into fine powder and stored in a sealed plastic bag. In the following step, 1 g of the crushed sample was combusted at 550 °C for 4–8 h using a Lenton kiln. The combustion did not stop until the samples completely turned white. The cooled samples were combined with a mixture of HNO₃:HCl (ratio 1:3) at a volume of 50 mL filtered and analyzed using the atomic absorption spectrometer machine (AA280FS, Varian, Australia) in the lab of the National Institute of Agriculture Planning and Projection.

Figure 2. Morphology of L. flava at harvest time.

Figure 2. Morphology of L. flava at harvest time.

The safety limit of Cd in edible parts was evaluated using the Vietnam National Technical Standards (QCVN8-2:2011/BYT) and the Food and Agricultural Organization/the World Health Organization (FAO/WHO), which is 0.05 mg/kg fresh weight (fw) for Cd.

2.4. Statistical Analysis

All the data obtained were statistically analyzed using a one-way analysis of variance (ANOVA) with the help of the SAS 9.1 software (SAS Institute Inc., Cary, NC, USA) to examine variations among the means at the probability level p < 0.05. In this analysis, the coefficients of variations, which represent the ratios of standard deviations to the means, were calculated and interpreted as an index of reproducibility and precision of the measurements. Least significant difference (LSD) was calculated at 5% probability for the treatments (T1–T4). Group Tukey SHD with alphabetic characters for modal mean values was used to compare differences with a 95% confidence interval in order to select the most effective treatment for the research’s purpose. The graphs were made by Origin Pro 2018 software (Northampton, MA, USA).

3. Results and Discussion

3.1. The Removal of Cd from the Soil

The analysis showed that the total and available Cd content slightly fluctuated during the experiments (

Table 1. Total Cd and flexible Cd in the soils before and after 60 days of the experiment (mg/kg dry soil).

Treatments (T)	Total Cd and Flexible Cd in the Soils (mg/kg Dry Soil)
	Total Cd		Reduction Rate(%)	Flexible Cd		Reduction Rate(%)
	Before Treatment	After Treatment		Before Treatment	After Treatment
T1	2.05 ± 0.187	2.0 ± 0.021	2.44	0.4 ± 0.178	0.35 ± 0.029	12.50
T2	5 ± 0.095	4.86 ± 0.022	2.8	1.15 ± 0.069	1.0 ± 0.034	13.04
T3	10 ± 0.09	9.56 ± 0.225	4.4	3.3 ± 0.026	2.86 ± 0.043	13.33
T4	20 ± 0.167	18.44 ± 0.215	7.8	11.2 ± 0.022	9.64 ± 0.029	13.93

).

Table 1 shows that a small amount of Cd was removed from the soils. In the T1 group, the total Cd content after the experiment showed a reduction of 2.44%. Meanwhile, the Cd concentrations added to the soil were 5, 10, and 20 mg/kg. The total Cd after the experiment varied between 4.86 and 18.44 mg/kg of dry, clean soil, equivalent to reductions of 2.80, 4.4, and 7.80%. Additionally, the available Cd content in the soil decreased significantly, indicating reductions of 12.50, 13.04, 13.33, and 13.93%, respectively. This result might have happened due to highly branched rootlet structures and complexes created by the plant microorganisms, and to the combination with flexible Cd, which lead to other non-toxic forms. Moreover, the total Cd and the flexible Cd were higher than the accumulation capability of L. flava plants in combination with the Penicillium chrysogenum strain fungi and Bacillus licheniformis bacteria [23,24]. Abhilash et al. [23] reported that the removal percentage of Cd by L. flava after 30 days of the treatment reached up to 93–98%. In addition, the findings reported by Deng et al. [24] indicated that the soil Cd contents after one- and two-step bioleaching using Penicillium chrysogenum were 0.63 and 0.82 mg/kg, respectively. The results prove that this combination significantly reduced the amount of Cd available in the soils.

3.2. The Accumulation of Cd in Microbial and Fungal Biomass

The amount of Cd accumulation in the biomass of microorganisms is shown in

Table 2. Ability to accumulate Cd in biomass of microbial and fungal strains.

Microbial Strains	Cd2+ Accumulation in Microbial Biomass in Different Concentrations
	1.5 mg/L	5 mg/L	10 mg/L	20 mg/L
Bacillus licheniformis	1.05 ± 0.04	2.85 ± 0.05	5.91 ± 0.13	34.70 ± 1.32
Penicillium chrysogenum strain	0.91 ± 0.04	7.87 ± 0.04	16.71 ± 0.09	33.43 ± 1.87

Note: Mean ± SD (n = 4) with the same letter (normal typeface) in the column and the same letter (capitalized) in rows indicates that values are not significantly different at p < 0.05 while different letters indicate the significant difference.

.

The levels of Cd²⁺ accumulation in Bacillus licheniformis biomass under soil Cd²⁺ concentrations of 2.05, 5, 10, and 20 mg/L were 1.05 ± 0.04, 2.85 ± 0.05, 5.91 ± 0.13, and 34.70 ± 1.32 mg/L, respectively. Meanwhile, the levels of Cd2+ accumulation in Penicillium chrysogenum biomass in similar conditions were 0.91 ± 0.04, 7.87 ± 0.04, 16.71 ± 0.09, and 33.43 ± 1.87, respectively. Under Cd²⁺ concentrations ranging from 2.05 to 10 mg/kg, Penicillium chrysogenum exhibited a higher Cd²⁺ accumulation capacity than Bacillus licheniformis. However, their Cd accumulation capacity trend tended to be roughly similar at high concentrations, equal to 20 mg/L. At low Cd levels, as low as 2.05 and 5 mg/kg in the soils, L. flava showed no signs of toxicity. Hence, L. flava is highly likely to absorb heavy metals and accumulate them in the plant cells [23]. Abhilash et al. [23] indicated that the removal efficiencies of Cd under concentrations of 0.5, 1, 2 and 4 mg/L by L. flava species were up to 98, 96, 95 and 93%, respectively.

Therefore, a low level of Cd concentration was accumulated in Penicillium chrysogenum and Bacillus licheniformis. At higher levels of Cd²⁺ (such as 10 and 20 mg/kg in the soils), the plant absorbed and accumulated a larger amount of Cd, exhibiting signs of plant toxicity through a decrease in height and biomass. Cd caused cellular modifications, such as growth inhibition, the alteration of cell morphology, and the generation of excessive reactive oxygen species, which affect the growth and reproduction of the plant and the microorganisms [25]. Baran and Duz indicated that the monolayer adsorption capacity of B. licheniformis for Cd (II) was found to be approximately 24.51 mg/g [26]. Similarly, Xu et al. [25] showed that the maximum removal of Cd (5 mM) by XJ-1 Penicillium chrysogenum fungi was approximately 86.21% in tightly bound Cd, 8.46% in intracellular-bound Cd, and 5.33% in loosely bound Cd. It should be noted that the cell walls of Penicillium chrysogenum fungi are rich in polysaccharides and glycoproteins that enable them to accumulate heavy metals [21]. Exopolysaccharide in the cells of B.licheniformis in combination with an anionic group can help to either absorb and/or remove heavy metals through an electrostatic interaction [27]. The effects of Penicillium chrysogenum and Bacillus licheniformis on the growth of some plants in soils with heavy metal stress were established by enhancing their ability to germinate seeds, such as in the case of rice plants under high values of Ni [15].

Previous studies on the application of the fungus Penicillium chrysogenum in combination with Brassicachinensis L. at different levels of CdCl₂ (e.g., 0, 1, 5, 10, and 50 mg/kg) in soils resulted in a higher yield of the Brassicachinensis L. plants, and increased their shoot biomass by 1.6–3.9 times compared to the T1 group without fungus. Although this fungus is not able to directly promote plant growth, it can invade the Cd-contaminated soils and reduce the bioavailability of Cd through biosorption. Therefore, the Cd uptake by the plants was reduced, enabling the plants to grow without signs of poisoning [25]. The preliminary selection of microorganisms proved that these two species were able to effectively resist Cd concentrations higher than 20 mg/kg.

3.3. The Growth of L. flava Plants under High Heavy Metal Concentrations

The heights and total dry biomass of the L. flava plants are shown in

Table 3. The growth of L. flava plants.

Treatments	T1	T2	T3	T4
Dry biomass (g/plant)	30.41 ± 0.7846	31.22 ± 0.3648	30.02 ± 0.4163	29.57 ± 0.5041
Height of plants (cm)	52.76 ± 0.5452	54.24 ± 1.0124	51.82 ± 1.421	49.95 ± 0.7816

.

When the concentrations of heavy metals in the soil surpass the critical resistance levels, the growth of the plants is affected, as represented in yellow leaves, along with a reduction in the height and plant biomass of the L. flava [23]. Nevertheless, plant biomass is one of the most important indicators to assess the accumulation potential of heavy metals by plants. The heights and total dry biomass of the plants in treatment T2 reached their highest values (54.24 cm and 31.22 g/plant). In the T1 group, the heights and total dry biomass of plants were ranked second highest among the other treatments, with 52.77 cm and 30.41 g/plants, respectively. In contrast, the heights and dry biomass of the plants were at the lowest values with 51.82 cm and 30.02 g/plants in treatment T3. These results were not significantly different in comparison with the treatments of T1 and T4, as shown in Figure 3a,b.

On the other hand, L. flava is an aquatic plant species growing with fixed roots which can survive in severe environmental conditions. This species is able to produce excessive biomass, harvest, and accumulate heavy metals [23]. This species was found to thrive strongly in agricultural soil highly contaminated with heavy metals, such as Pb, Cd, As, and Hg in the Mekong Delta, located in the southeast of Vietnam [28]. The study on L. flava combined with microbial inoculants demonstrated that L. flava exhibited a normal growth in stressed soil conditions containing different levels of Cd (between 2 and 20 mg/kg). These effects should be evaluated not only through considering the Cd tolerance of L. flava, but also based on the role of Bacillus licheniformis and Penicillium chrysogenum in their mutually symbiotic relationships with L. flava in the soil and root zones. With the added microbial inoculants, the increase in microorganisms and fungi enhanced plant growth, either directly or indirectly, through a variety of mechanisms, such as allowing plants to develop longer roots during early growth stages in order to reduce ethylene production [15,29]. Park et al. [15] indicated that the Bacillus licheniformis MH48 rhizobacterium could enhance the nutrient uptake of the seedlings of the ornamental plant Camellia japonica (Saemangeum, Korea). These microorganisms exist in the soils and facilitate metabolic processes in order to produce organic acids, which is one of the mechanisms to solubilize the phosphorus (P) attached to insoluble mineral compounds in soil [15].

The microorganisms and fungi utilize this process for nitrogen fixation, promoting specific enzymatic activity and supplying bioavailable phosphorous as well as altering trace elements that enhance the capacity of plants to uptake nutrients [29]. Javed et al. [29] reported that Penicillium chrysogenum can increase the growth of plants to their highest level through its phosphate solubilizing ability. The production of phytohormones in microorganisms and fungi such as auxins, cytonins, and gibberellins increases the tolerance capacity and growth ability of L. flava in high Cd concentrations [29,30]. The microbes synthesize biologically active compounds, including phytohormones and antifungal compounds, which play a vital role in plant growth, nutrition, and development [30]. Baciluss bacterium is a rhizospheric microbiome that facilitates the production of siderophores and is a crucial element in solubilizing unavailable forms of heavy metal deposits through complexation reactions [31]. Consequently, Penicillium chrysogenum, Bacillus licheniformis, and L. flava had joint effects on Cd removal in the soils. It was found that an increase in plant biomass and the growth of fungi and microorganisms had close relationships to Cd accumulation in the plant particles. Hence, the nutrients, moisture, and pH levels need to be adjusted to their optimum values to enable the development of plants, bacteria, and fungi, thereby increasing the removal of Cd from the soils [15,29,30].

3.4. The Accumulation Capacity of Cd by L. flava Plants

The accumulation capacity of Cd by L. flava plants is shown in

Table 4. The accumulation capacity of Cd by L. flava plants.

Treatments	T1 (mg)	T2 (mg)	T3 (mg)	T4 (mg)
Inedible part	0.634 ± 0.02186	1.643 ± 0.02093	3.32325 ± 0.02750	3.32325 ± 0.01435
Edible part	0.01 ± 0.00216	0.031 ± 0.00365	0.05 ± 0.00258	0.4305 ± 0.00265

.

At different levels of Cd in soil, including 2.05, 5, 10, and 20 mg/kg, with similar levels of microbial inoculants, the accumulation of Cd in inedible parts (roots, stems, and leaves) was recorded. The accumulation of heavy metals in plants is considered to be an important indicator for assessing each plant’s potential to remove heavy metals as well as the combined effects of microbial inoculants and plant growth on contaminated soils. These findings show that the plant growth gradually increased the Cd accumulation in the inedible parts (roots, stems, and leaves) of L. flava plants in the respective treatments (T2–T4) in comparison with the T1 group at the 95% significance level. In this respect, the concentration of Cd added to the soil (e.g., 20 mg/kg) reached the highest accumulation level (6.81 mg/kg dry weight) in the inedible parts of the plant (group T4) compared to the control group (T1), which was only 0.63 mg/kg dry weight. The second highest increase was observed in the T3 group (3.32 mg/kg dry weight), which was 5.27 times higher than the T1 group. The accumulation was lowest in T2 at 5 mg/kg Cd concentration (1.64 mg/kg dry weight), only 2.6 times higher than the T1 group (Figure 4).

The application of the microbial inoculants, Bacillus Licheniformis and Penicillium Chrysogenum, developed on the root zone of L. flava increased the absorption surface of the roots, helping the roots to absorb large amounts of Cd accumulated in the mycelium as well as on the surface of the bacterial cell walls [32,33]. In turn, these microorganisms usually capture, bind, and transfer Cd ions into a flexible form that is bioavailable for the roots. This result was demonstrated by a previous study [32] which indicated that the bioaugmentation of the rhizosphere of Indian mustard with a high density of Bacillus Licheniformis increased the accumulation of Cd in the tissues of the plant. Deng et al. [34] studied Penicillium chrysogenum F1 for the bioleaching of heavy metals from contaminated soils and showed that 19.8 mg of Cd, Cu, Pb, and Zn could be extracted from 2.5 g of soil, while the total biological leaching ratio of the heavy metals was 60.4%.

The activity of these microorganisms at high Cd concentrations in soils promotes phosphorus resolution, which helps plants to absorb nutrients and improves the plant growth. All of these factors are likely to contribute to an increase in the ability of the roots, stems, and leaves of L. flava to accumulate Cd [15,30]. Cd is a non-essential element that is highly mobile in organisms in which, following plant uptake, subcellular distribution plays an important role in plant tolerance and detoxification [35]. L. flava is commonly used to treat pollution in environments contaminated with heavy metals. This species was used to remove Cd in Indian wetlands and promote Cd phytofiltarrtion in contaminated waters [24].

3.5. Assessment of Cd Accumulation in the Edible Parts of L. flava

The combination of microbial inoculants with L. flava at different concentrations of Cd affected the absorption and accumulation of Cd in the leaves and stems of L. flava.

The results showed that the accumulation of Cd content in the fresh weight of flowers, flower peduncle, and flower buds significantly increased at p < 0.05 and increased proportionally with the Cd concentration in the soils. The Cd content accumulated at the lowest level in the background concentration (0.01 mg/kg of fresh weight), whereas the highest Cd accumulation was 0.431 mg/kg in the fresh weight in T4 (Figure 5). The amount of Cd accumulation in the edible parts (0.01 and 0.031 mg/kg of fresh weight) were within the safety limit threshold under the QCVN 8-2: 2011/BYT and the FAO/WHO. However, regarding the added Cd concentration, the Cd accumulation levels in the edible parts were 1.28 times and 8.6 times higher than the maximum permissible safety limit (0.05 mg/kg), as specified by the aforementioned organizations.

This result was consistent with the observation reported by Abhilash et al. [23], indicating that the accumulation of Cd in L. flava roots was higher than that of leaves, stalks, and flowers in four treatments (0.5, 1, 2, and 4 mg/L) within 30 days. The hyphae of Penicillium chrysogenum grew on the roots, creating contact, and spread widely over the soil [23,24]. As a result, a large amount of Cd remained in the mycelium structure of the roots and spores, resulting in a large reduction of the Cd added to the soil, and increasing the possibility of Cd accumulation in the flower parts. Therefore, microbial products can be used in combination with L. flava in highly Cd-contaminated areas (e.g., with Cd < 10 mg/kg) for growing commercial vegetables, but they should not be grown in seriously polluted areas because of the high risk of toxicity. Thus, applications of microbial inoculants with L. flava for treating Cd contamination in soil should be limited to areas with lower than 10 mg/kg, in accordance with the QCVN 03-8: 2015/BTNMT. The mean value of Cd concentration in the soil must be around the range of ≤7.5 mg/kg. The observations of this study indicate that farmers can grow multi-purpose L. flava plants to clean Cd-contaminated agricultural soil in the range of ≤7.5 mg/kg.

The Cd accumulation in the edible parts of L. flava plants in soil containing heavy metal in T1 (2.05 mg/kg) and T2 (5 mg/kg) was below the safety limit threshold under the QCVN 8-2: 2011/BYT and the FAO/WHO, which is safe to use in human food. In contrast, in the case of soil containing heavy metals in T3 (10 mg/kg), the Cd accumulation in the edible parts of L. flava was barely over the safety limit threshold, which indicates it can be used for animal consumption. In T4 (20 mg/case), the Cd accumulation in the edible parts of L. flava exceeded the allowable limit for collection in a centralized treatment or landfill by more than eight times, according to the processing procedure of enviromental government agencies.

3.6. Correlation Matrix

Figure 6 shows the correlation matrix between plant height (PH), biomass (BM), edible (ED) parts, and inedible (IED) parts by L. flava. The results indicated that the inedible plant parts have positive correlation with the edible parts of L. flava. However, plant height was negatively significantly correlated with the inedible and edible parts of the L. flava plant. In addition, the biomass of L. flava was negatively correlated with the inedible and edible parts of the L. flava plant, but positively correlated with plant height.

The correlation matrix analysis revealed that ED was 0.95 positively correlated with IED. Meanwhile, PH had a negative −0.83 correlation with IED and −0.86 with ED, respectively. However, BM was observed to be negatively correlated −0.61 with IED and −0.62 with ED, whereas the positive correlation among BM was noted as 0.93 with PH. It was observed that an increase in Cd containment by L. flava had a negative impact on ED and PH. The Cd accumulation could affect nutrient uptake and the photosynthetic performance in L. Flava plants [36]. Sun et al. [36] indicated that decreases in K+ concentration in Cucumis sativus stems were observed to be 7.4, 14.1, 26.9, 38.9, and 48.8% with the application of 10, 25, 50, 100, and 200 µM Cd treatments. The Cd contained in IED and ED could inhibit plant growth by decreasing the water-use efficiency and the net rate of photosynthesis in L. flava. The results revealed that the Cd remained in L. flava restricted the development of the plant. Bahadur et al. [37] observed a positive correlation among Cd content in maize shoots and Cd in heavily and mildly polluted soils.

4. Conclusions

Microbial inoculants containing a mixture of two selected strains of microorganisms belonging to the Penicillium Chrysogenum fungus and the Bacillus Licheniformis bacterium were grown on agricultural soil artificially contaminated with Cd (concentrations of 2.05 (background), 5, 10, and 20 mg/kg). The incorporation of these microorganisms did not adversely affect the growth, biomass, or commercial yield of the respective plants. Meanwhile, the plants were best stimulated at a concentration of 5 mg/kg Cd added to the soil. The mixed infection of the two selected strains of microorganisms in the form of inoculants increased the accumulation of Cd in parts of the L. flava plants in proportion to the concentration of the Cd added to the soil. L. flava could be grown on agricultural soil contaminated with Cd at concentration levels lower than ≤5 mg/kg.