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Article

Evaluation of the Effectiveness of Bioaugmentation-Assisted Phytoremediation of Soils Contaminated with Petroleum Hydrocarbons Using Echinacea purpurea

by
Katarzyna Wojtowicz
*,
Teresa Steliga
* and
Piotr Kapusta
Oil and Gas Institute—National Research Institute, ul. Lubicz 25 A, 31-503 Krakow, Poland
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(24), 13077; https://doi.org/10.3390/app132413077
Submission received: 30 October 2023 / Revised: 6 December 2023 / Accepted: 6 December 2023 / Published: 7 December 2023
(This article belongs to the Special Issue Environmental Pollution and Bioremediation Technology)
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The results obtained during the study of phytoremediation assisted by bioaugmentation will in the future contribute to a better and faster process of cleaning areas contaminated with petroleum substances.

Abstract

Phytoremediation supported by bioaugmentation is a promising method considered for cleaning up areas polluted with petroleum hydrocarbons. In this study, phytoremediation was carried out using Echinacea purpurea as a phytoremediant on two types of soil: Soil DW—aged soil taken from an excavation pit, Soil OS—soil taken from an oil spill area. The tests for each soil were carried out in six test systems (non-inoculation, inoculation with the B1 microbial consortium, inoculation with the B2 microbial consortium, inoculation with the B1 microbial consortium with the addition of γ-PGA (γ-poly glutamic acid), inoculation with the B2 microbial consortium with the addition of γ-PGA and inoculation with the γ-PGA solution) for 6 months. The effectiveness of the remediation treatments used was assessed based on chromatographic analyses of soil and plant material (roots, shoots) and toxicological analyses using four types of toxicological tests (PhytotoxkitTM (MicroBioTests Inc., Gent, Belgium), OstracodtoxkitTM (MicroBioTests Inc., Gent, Belgium), Microtox® Solid Phase Test (Modern Water Inc., New Castle, DE, USA), MARA (NCIMB Ltd., Aberdeen, UK)). The research conducted showed that the most effective method of bioremediation of soils contaminated with petroleum hydrocarbons was phytoremediation supported by bioaugmentation with the microbial consortium B2 with γ-PGA, which allowed for reducing the concentration of total petroleum hydrocarbons (TPHs) and polycyclic aromatic hydrocarbons (PAHs) in the tested soils by 53.98% and 49.54% (Soil DW-5) and 60.47% and 37.55% (Soil OS-5), respectively. In turn, the lowest bioremediation efficiency was recorded in non-inoculated systems, for which the concentration of TPHs and PAHs at the end of the study decreased by 18.40% and 16.14% (Soil DW-1) and 21.87% and 18.20% (Soil OS-1), respectively. The results of toxicological analyses confirmed the relationship between the concentration of TPHs and PAHs in the soil and its toxicity level.

1. Introduction

The soil environment is prone to contamination by both organic and inorganic substances, which poses potential threats to the fauna and flora. Contamination of soils with petroleum hydrocarbons and heavy metals with carcinogenic, mutagenic and teratogenic properties seems particularly dangerous [1,2]. In view of the above, there is an urgent need to rehabilitate the areas contaminated with such xenobiotics and restore them [3]. One common method of land remediation is phytoremediation, which is a green chemistry technique, which uses plants capable of removing, metabolizing, assimilating and adsorbing harmful substances. Plants used in phytoremediation are classified as so-called hyperaccumulators. Hyperaccumulators are plants characterized by their ability to accumulate large amounts of xenobiotics without the harm caused by phytotoxic effects [4,5,6]. For sites contaminated with petroleum hydrocarbons, plants belonging to the so-called naphthophytes are used in the phytoremediation process. Naphthophytes show an evolutionarily acquired ability to grow on oil lands [7,8,9]. Identifying potential plants, which can be used in the remediation of land contaminated with both organic and inorganic substances, is an interesting area of research into the phytoremediation process. One promising plant in this regard appears to be purple coneflower (Echinacea purpurea).
Echinacea purpurea, commonly known as purple coneflower, is a plant species from the Asteraceae family, originating from North America [10]. Adaptability to different climatic conditions, resistance to environmental stress and the ability to grow in different soil conditions have made Echinacea purpurea suitable for cultivation in most climate zones. In addition, Echinacea purpurea is characterized by high biomass production and a well-developed root system (fibrous root). The large surface area and high activity of the root system (production of root exudates) of purple coneflower allow for the stimulation of microbial activity in the root zone, thus enhancing mineralization at the root–soil interface, which enables rhizodegradation [11]. Among other mechanisms occurring during phytoremediation are biodegradation, phytoextraction, phytovolatilization, phytostabilization and abiotic loss, which can act individually or co-occur [12]. Phytoremediation studies conducted using Echinacea purpurea have shown that the plant has a high tolerance for petroleum and is suitable for remediating soils with petroleum concentrations exceeding 10,000 mg/kg [11]. In addition, purple coneflower has been proven to be effective in cleaning up soils polluted with PAHs and alkyl-PAHs [13,14,15,16]. A few studies also suggest that Echinacea purpurea can be used to clean up areas tainted with heavy metals [13,14]. However, it should be noted that when planning remediation strategies for land contaminated with various types of xenobiotics, the concentration, age and type of contamination should be taken into account. It has been proven that aged soils containing a greater variety of contaminants, with higher concentrations of xenobiotics, undergo the treatment process more slowly than freshly contaminated soils with a single type of contaminant. For real soils with a wide spectrum of contaminants, the efficiency of phytoremediation alone may be too low [17]. Therefore, it is critical to find a new, efficient and economically beneficial approach to the phytoremediation process.
In recent years, so-called combined methods using micro-organisms have become increasingly popular for remediating land contaminated with petroleum substances [18]. The most commonly used combination methods include phytoremediation in combination with bioaugmentation. It is based on the interaction of plants and microbes to increase the efficiency of pollutant degradation. The bioaugmentation-assisted phytoremediation method exploits the potential of micro-organisms present in the inoculant to biodegrade petroleum pollutants and their influence on the phytoremediation process. Increasing the concentration of bacteria, fungi and yeast in the root zone has been proven to promote root growth, allowing it to extract more nutrients from the soil, resulting in better growth. Moreover, the symbiotic interaction of micro-organisms with the root system allows for a better degradation of pollutants, transformation of complex organic molecules into simpler and less toxic compounds and their transfer to the plant [18,19,20,21]. It should be remembered that the inoculant used in bioaugmentation treatments should contain micro-organisms capable of degrading a broad spectrum of contaminants. The bacteria most commonly described in the literature for use in remediating land contaminated with petroleum hydrocarbons include Achromobacter sp. [22,23], Bacillus sp. [23,24,25,26,27,28,29], Dietzia sp. [30,31], Gordonia sp. [32,33], Mycolicibacterium sp. [34,35,36,37,38,39], Pseudomonas sp. [40,41,42,43,44,45], Rhodococcus sp. [46,47,48,49,50,51]. Among the fungi capable of metabolizing oil-derived substances are Aspergillus sp. [52,53], Candida sp. [54,55], Cladosporium sp. [56,57], Fusarium sp. [58,59] and Penicillium sp. [60,61]. It should also be remembered that in order to ensure the best possible efficiency of the bioaugmentation process, it is advisable to use inoculants developed on the basis of selected micro-organisms isolated from the natural environment. This avoids antagonistic interactions with naturally occurring micro-organisms in the tested soil [17]. In addition, substances can be added to the inoculant to ensure better microbial growth by providing an easily assimilable carbon source or using a natural biosurfactant, such as γ-PGA [3,17,62,63].
Bioaugmentation and phytoremediation are technologies, which have been studied individually many times, but there are few publications describing their interaction in the bioremediation of soils polluted with TPHs and PAHs. It was assumed that the correct choice of inoculant in the bioaugmentation-assisted phytoremediation method would help ensure high efficiency of the treatment process of contaminated land. In the present study, it was decided to experimentally test the effectiveness of the combination of phytoremediation and bioaugmentation in several variants of conducting the process on real samples of land contaminated with TPHs and PAHs, and to evaluate the change in toxicity of soils after treatment.

2. Materials and Methods

2.1. Soil, Microbial Consortia, γ-PGA and Plant Description

Two types of soil contaminated with petroleum hydrocarbons were used in the study. Soils were taken from two locations, which were the G-70 pit area—which was used in the 1970s to store drilling waste mixed with soil (Soil DW)—and an oil spill site (Soil OS). Forty soil samples weighing 2 kg were collected from each site. Samples were taken from the surface layer (0–0.25 m below ground level) and the deep layer (0.25–1.5 m below ground level). Each type of soil taken was mixed and homogenized for averaging, followed by physical and chemical analyses. The composition of the soils used in the bioaugmentation-assisted phytoremediation study is shown in Table 1.
Two microbial consortia developed from indigenous micro-organisms isolated from contaminated sites were used to carry out the bioaugmentation procedure. Microbial consortium B1 contained the bacterial strains Dietzia sp. IN118, Gordonia sp. IN101, Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus glopberulus IN113 and Raoultella sp. IN109. Microbial consortium B2 consisted of bacteria present in microbial consortium B1 and was additionally enriched with fungal strains Aspergillus sydowii, Aspergillus versicolor, Candida sp., Cladosporium halotolerans, Penicillium chrysogenum. The density of cells in both consortia was in the range of 5 × 108–1 × 109 CFU/mL. The bacterial and fungal strains used in the microbial consortia had proven abilities to metabolize petroleum hydrocarbons and did not exhibit pathogenic properties. A detailed description of the design of microbial consortia B1 and B2, along with the method of identification of the strains of bacteria and fungi present in them, is provided in a previous article [17]. More information about the microbial consortia used in the research is available in Supplementary Materials. A commercially available formulation of Ambiogel® (Ambioteco, Staszów, Poland) containing 5% pure poly-γ-glutamic acid [64], whose impact on the efficiency of bioremediation has been confirmed in earlier studies, was used as an additive for microbial consortia. The phytoremediation process was carried out using seeds of Echinacea purpurea (Legutko, Poland), which were sown in each of the tested systems.

2.2. Course of the Experiment

Phytoremediation studies using Echinacea purpurea as a phytoremediator and bioaugmentation-assisted phytoremediation were conducted under laboratory conditions using the vase method. Experiments were conducted on two types of soil with known contaminant concentrations. Prior to soil treatment, the soil was fertilized with the mineral fertilizer “Azofoska” (Inco, Warszawa, Poland) composed of 13.6% N-total nitrogen; 5.5% N-nitrogen; 8.1% N-ammonium nitrogen; 6.4% P2O5 soluble in neutral ammonium citrate solution and water, 4.2% P2O5 soluble in water; 19.1%K2O soluble in water; 4.5% MgO; 23.0% SO3; 0.045% B; 0.18% Cu; 0.17% Fe; 0.27% Mn; 0.04% Mo; 0.045% Zn. This was to ensure that the C:N:P ratio in the tested soils was 100:10:1 [1,17,35], which is optimal for the proper development and activity of micro-organisms responsible for hydrocarbon degradation. The soil reaction was brought to a value of 7.5–7.6 through supplementation with fertilizer lime of natural origin (Biovita, Tenczynek, Poland) [65,66,67]. Soils prepared in this way were placed in 10 L pots, and Echinacea purpurea was sown in them. The process of treating contaminated soils was carried out in 12 systems. Depending on the system studied, the soil was bioaugmented with the appropriate inoculant. Systems 1 and 7—soils not inoculated, systems 2 and 8—soils inoculated with microbial consortium B1, systems 3 and 9—soils inoculated with microbial consortium B2, systems 4 and 10—soils inoculated with microbial consortium B1 with the addition of γ-PGA, systems 5 and 11—soils inoculated with microbial consortium B2 with the addition of γ-PGA, systems 6 and 12—soils inoculated with γ-PGA. The study was carried out in two series of repetitions in a greenhouse, ensuring constant conditions for the experiment, i.e., temperature of 22–25 °C, aeration, humidity (soil sprinkling) and sunlight (system of LED lighting lamps with a light intensity of 3000–5000 lumens ensuring the maintenance of the day and night cycle). The first series of studies was completed after 3 months, while the second series was completed after 6 months. This allowed us to control changes in the amount of TPHs and PAHs in soil and plant material during the treatment process carried out in the systems studied. A schematic of the test bed for bioaugmentation-assisted phytoremediation is shown in Figure 1.
After the experiment, the test plants were removed from the pots in which phytoremediation tests were carried out and then washed with distilled water to remove any soil residues. Plants were separated into shoots and roots. The plant material was dried and homogenized, and its biomass was determined. After testing, the soil was dried to an air-dry state, ground and sieved through a 1 mm sieve. Soil samples and plant material prepared in this way were analyzed for TPH and PAH content. In addition, the soil was tested for changes in toxicity using commercially available toxicological tests.

2.2.1. Analysis of TPH in Soil and Plant Material

Extraction of TPH from the soil/plant material was carried out by sonication. For this purpose, the dried test material (5 g) was placed in a 200 mL Erlenmajer flask, followed by the addition of 20 mL of dichloromethane (Chempur, Piekary Śląskie, Poland). The analyte extraction process was carried out in three repetition series of 15 min each. The resulting extracts were filtered and then subjected to purification of polar substances by dSPE solid-phase extraction. Purification of the extracts was carried out on pre-conditioned Florisil Chromabond columns No. 730081 (Macharey-Nagel, Dueren, Germany). The purified extracts were concentrated on a rotary evaporator (Chemland, Piekary ŚląskiePoland) and subjected to chromatographic analysis using a Clarus 500 gas chromatograph from Perkin Elmer (Waltham, MA, USA), according to the procedure for determining TPH developed at the Department of Complex Fluids Exploitation Technology at INiG—PIB [65,66]. The most important operating parameters of the chromatograph are listed in Table 2.

2.2.2. Analysis of PAHs in Soil and Plant Material

Isolation of PAHs from soil/plant material was carried out using the QuEChERS method developed at the Department of Reservoir Fluid Exploitation Technology at INiG—PIB (oil–gas). The dried test material was placed in a 50 mL vial, followed by the addition of 5 mL of distilled water, 10 mL of acetonitrile (Chempur, Piekary Śląskie, Poland) and MgSO4+ NaCl extraction mixture No. SST640 (Interchim, Montluçon, France). The vials were tightly sealed and shaken for 5 min. After this time, the samples were transferred to an MPW-223e centrifuge (MPW, Warszawa, Poland) and centrifuged at 3500 rpm for 10 min. The decanted extract was cleaned of interfering substances in special Clean-up Kit Fruit and Vegetables AOAC vials No. JO3937 (Interchim, Montluçon, France). Purified extracts were filtered through a syringe filter with a 0.2 μm nylon (PA) membrane and subjected to chromatographic analysis. PAH determination was performed on a Vanquish Core Series liquid chromatograph from Thermo Fisher Scientific (Waltham, MA, USA) [17,68]. The most important operating parameters of the chromatograph are listed in Table 2.

2.2.3. Soil Toxicity Analysis

The toxicity of soils at baseline and after completion of the bioaugmentation-assisted phytoremediation process carried out in the studied systems was evaluated using 4 commercially available microbiotests: PhytotoxkitTM (MicroBioTests Inc., Gent Belgium), OstracodtoxkitTM (MicroBioTests Inc., Gent, Belgium), Microtox® Solid Phase Test (Modern Water Inc., New Castle, DE, USA) and MARA (NCIMB Ltd., Aberdeen, UK) [65]. PhytotoxkitTM and OstracodtoxkitTM tests are among the chronic toxicity assessment tests. The PhytotoxkitTM test is based on evaluating the germination and early growth of test plants, which include Sorghum saccharatum, Lepidium sativum and Sinapis alba. The result of the test is inhibition of germination and early root growth after three days of incubation at 25 °C. The OstracodtoxkitTM test is based on assessing the percentage mortality and growth inhibition of crustaceans of the species Heterocypris incongruens after 6 days of contact with the test soil. The Microtox® Solid Phase Test is an acute toxicity test and involves evaluating the decrease in fluorescence of Vibrio fischeri bacteria following contact with the test sample. The result of the Microtox® Solid Phase test is EC50, which is the concentration of the test sample, which produces 50% of the test reaction. To assess environmental risk, the MARA test was used, based on evaluating the degree of growth inhibition of 18 test organisms (Microbacterium spaciec, Brevundimonas diminuta, Citrobacter freudii, Comamonas testosteroni, Entrococcus casseliflavus, Delftia acidovorans, Kurthia gibsoni, Staphylococcus warneri, Pseudomonas aurantiaca, Serriatia rudidaea and Pichia anomala) after 18 h of incubation [17,62,65,69]. All toxicological tests were conducted in accordance with the manufacturers’ procedures, while the results obtained were converted to TU values.

3. Results

3.1. Analysis of Petroleum Hydrocarbons in Soil, Root and Shoot

Chromatographic analyses of the soil for TPH content showed that bioaugmentation with microbial consortia developed on the basis of indigenous micro-organisms significantly affects the efficiency of its treatment process using Echinacea purpurea as a phytoremediator. At the end of the 6-month experiment, in non-inoculated systems, TPH biodegradation efficiency was low, at 18.40% (Soil DW-1) and 21.87% (Soil OS-1). The application of inoculation with bacterial microbial consortium B1 increased the biodegradation rate of TPH to 35.56% (Soil DW-2) and 40.85% (Soil OS-2). In systems inoculated with microbial consortium B2 enriched with fungal species capable of degrading petroleum hydrocarbons, TPH biodegradation rates were 44.16% (Soil DW-3) and 49.78% (Soil OS-3). In addition, it was observed that the use of γ-PGA significantly increases the efficiency of cleaning up soils polluted with petroleum substances. In soil taken from the site of an oil spill pit (Soil DW), the application of inoculation with microbial consortia B1 and B2 with the addition of γ-PGA in the phytoremediation process resulted in a reduction in TPH concentrations by 46.71% in system 4 and 53.98% in system 5. In addition, in the system inoculated with γ-PGA alone, phytoremediation efficiency using Echinacea purpurea was 3.4% higher than in the non-inoculated system. Similarly, in soil taken from an oil spill site (Soil OS), the phytoremediation efficiency was highest in systems inoculated with microbial consortia containing γ-PGA, achieving 51.19% (Soil OS-4) and 60.47% (Soil OS-5). Inoculation with pure γ-PGA compared to the non-inoculated system led to an increase of 5.02%. In addition, based on chromatographic analyses using two types of soil with different ages of contamination, it was found that aged soils (Soil DW) were less amenable to treatment than freshly contaminated soils (Soil OS). The changes in TPH concentrations in DW and OS soils subjected to bioaugmentation-assisted phytoremediation in the systems studied are shown in Figure 2.
For a complete evaluation of the effectiveness of the bioaugmentation-assisted soil phytoremediation process, the distribution of alkanes in the studied systems before and after the experiment was determined. It was determined that hydrocarbons with a carbon chain length of nC10–nC21 were best removed from the soil matrix. Slightly worse values of biodegradation degrees were obtained for light hydrocarbons with carbon chain lengths of nC6–nC9. Heavy hydrocarbons with carbon chain lengths of nC22–nC30 and nC31–nC36 degraded at a much lower rate, consistent with previous studies. Moreover, it was observed that in soils vaccinated with a microbial consortium enriched with selected fungal species, a greater reduction in the content of heavy hydrocarbons was obtained than in systems inoculated with a bacterial biopreparation. A summary of the concentrations of selected groups of aliphatic hydrocarbons in DW and OS soils before and after phytoremediation studies in different inoculation systems is provided in Table 3.
Chromatographic analyses of the roots and shoots showed that aliphatic hydrocarbons can be extracted from the soil into Echinacea purpurea in all systems tested. However, it was observed that in systems not inoculated and inoculated with γ-PGA alone, the concentration of TPH in the roots and shoots was very low, which directly affected the efficiency of phytoremediation. In addition, poor growth of Echinacea purpurea was observed in systems 1 and 7, and 6 and 12 due to a poorly developed root system. Low TPH concentrations are also due to the fact that the diffusion of petroleum substances into the plant is greatly impeded due to the hydrophobic nature of hydrocarbons and their large molecular weights. Therefore, the uptake of lipophilic substances by plants is positively correlated with the lipid content of the root epidermis. Bioaugmentation of the soil with microbial consortia B1 and B2 allowed for the concentration of micro-organisms to be increased in the root zone of the phytoremediant used, which had a beneficial effect on the development of roots, and thus, the growth of Echinacea purpurea. In addition, it is likely that soil bioaugmentation increased the plant’s production of surface-active compounds, such as small-molecule organic acids or polypeptides, capable of lowering surface tension and breaking down or converting complex organic compounds into simpler compounds with greater bioavailability [8,9,70,71,72,73,74]. Therefore, higher concentrations of TPH were determined in the analyzed plant material taken from systems inoculated with B1 and B2 microbial consortia. Moreover, the use of γ-PGA as an additive to microbial consortia made it possible to provide the plant with a readily available source of carbon and energy, which further influenced biomass production. It is also worth noting that some literature sources report that γ-PGA can be considered a biosurfactant, which affects the diffusion of xenobiotics into the root [17,62,64,75]. Chromatographic analyses of the roots and shoots showed that extraction of aliphatic hydrocarbons from the soil to the root occurred most readily in systems 4 and 5, and 10 and 11. It should also be noted that in all systems tested, higher concentrations of TPH were obtained in the roots of Echinacea purpurea than in the shoots. A summary of TPH concentrations in the roots and shoots of Echinacea purpurea as a function of time and the system tested is provided in Figure 3 and Figure 4.
Chromatographic analyses of the soils for polycyclic aromatic hydrocarbons confirmed the observations made during TPH analyses. The PAH concentration in systems not inoculated and inoculated with γ-PGA alone was the lowest, at 16.14% in Soil DW-1, 14.19% in Soil DW-6, 16.28% in Soil OS-1 and 18.20% in Soil OS-6. Phytoremediation assisted by bioaugmentation with B1 and B2 microbial consortia reduced PAH concentrations in the tested soils by 30.82% (Soil DW-2), 36.73% (Soil DW-3), 27.10% (Soil OS-2) and 31.07% (Soil OS-3). The use of γ-PGA as an additive to microbial consortia enabled an additional reduction in PAH concentrations in the tested soils, with biodegradation rates of 39.22% (Soil DW-4), 49.54% (Soil DW-5), 32.89% (Soil OS-4) and 37.55% (Soil OS-5). The changes in ΣPAH concentrations in SD and OS soils subjected to bioaugmentation-assisted phytoremediation are shown in Figure 5.
The tested soils significantly differed in the qualitative and quantitative composition of individual groups of polycyclic aromatic hydrocarbons. Soil taken from the pit area (Soil DW) had higher concentrations of naphthalene relative to the other PAHs. In contrast, in the soil taken from the oil spill site, the distribution of the different groups of PAHs was more even, with a slight predominance of three- and four-ring PAHs. In such case, it is extremely important to study the distribution of individual groups of polycyclic aromatic hydrocarbons, taking into account the number of hydrocarbon rings. Indeed, the efficiency of biodegradation and phytoremediation depends on the number of rings in the hydrocarbon molecule [17]. Qualitative and quantitative chromatographic analyses of PAHs showed that naphthalene was most readily degraded in both tested soils. Lower values of biodegradation coefficients were recorded for three-ring PAHs. Four-ring PAHs were definitely less biodegradable. Six-ring PAHs had by far the worst distribution. It can therefore be assumed that during phytoremediation, bioaugmentation-assisted PAHs are degraded in the following order: PAH-2-ring → PAH-3-ring → PAH-4-ring → PAH-5-ring → PAH-6-ring [17,62,76]. Analyzing the qualitative and quantitative composition of the treated soils, it was observed that despite the higher degrees of biodegradation of the sum content of polycyclic aromatic hydrocarbons in SD-1-6 soils compared to the similarly inoculated OS-1-6 soils, the degrees of biodegradation of individual PAH groups were higher in OS-1 soils. This is influenced by the qualitative and quantitative composition of PAHs and the age of the pollutants. Considering the method of bioaugmentation-assisted phytoremediation, it can be seen that the use of fungi in the microbial consortia and the addition of γ-PGA positively affect the biodegradation efficiency of PAHs in soils. A summary of the concentrations of selected groups of polycyclic aromatic hydrocarbons in DW and OS soils before and after phytoremediation studies in different inoculation systems is provided in Table 4.
Chromatographic analyses of Echinacea purpurea roots and shoots in the tested systems showed insignificant concentrations of the analyzed xenobiotics in plant tissues. As with TPH, the highest PAH concentrations were determined in plants growing in systems inoculated with microbial consortium B1 and B2 with γ-PGA. Slightly lower PAH values were determined in the roots and stems of Echinacea purpurea taken from systems inoculated with microbial consortium only. In plants taken from non-inoculated systems and those inoculated with γ-PGA alone, the PAH concentrations were trace. Regardless of the system tested, higher amounts of PAHs were determined in root tissues than in shoot tissues. A summary of PAH concentrations in the roots and shoots of Echinacea purpurea as a function of time and the system tested is provided in Figure 6 and Figure 7.

3.2. Toxicological Analysis

Toxicological tests carried out on initial soil samples and after remediation treatments in the studied systems allowed for the assessment of the impact of changes in the concentrations of petroleum substances on soil biocenosis. Each of the toxicological tests performed showed the greatest decrease in soil toxicity in systems in which phytoremediation (Echinacea purpurea) was carried out supported by bioaugmentation with the B2 microbial consortium with the addition of γ-PGA. Phytoremediation supported by vaccination with the B1 microbial consortium with γ-PGA turned out to be slightly less effective. By far the smallest decrease in soil toxicity was observed in non-inoculated systems and in systems inoculated with γ-PGA alone. The obtained results of toxicological tests were consistent with the results of chromatographic analyses, which confirms the assumption that soil toxicity largely depends on the amount of TPHs and PAHs in the soil. A summary of the results of all toxicological tests performed for Soil DW and Soil OS subjected to the bioremediation process in the tested systems is presented in Figure 8 and Figure 9.
Manufacturer-level toxicity tests carried out using the Phytotoxkit test took into account two toxicity assessment criteria: inhibition of root growth and inhibition of germination. Taking into account the criterion of root growth inhibition, the toxicity expressed in the TU toxicity unit was determined for Soil DW and Soil OS initial soils at the levels of 12.2 and 9.8 (Lapidum sativum), 10.5 and 8.5 (Sorgum saccharatum), and 12.3 and 9.2 (Sinapis alba), respectively. The degrees of toxicity of Soil DW after bioremediation in the tested systems ranged from 10.5 to 4 (Lapidum sativum), 9.8 to 3.6 (Sorgum saccharatum) and from 10.3 to 3.3 (Sinapis alba). The toxicity of Soil OS after 6 months of purification, depending on the tested system, was in the range of 9.8–3.7 (Lapidum sativum), 7.8–3.3 (Sorgum saccharatum) and 9.5–2.9 (Sinapis alba). For the second parameter tested (germination inhibition), the TU values calculated for the tested plants in the initial soil samples were in the range of 9.3–7.6 (Soil DW) and 6.8–5.3 (Soil OS). As a result of the remediation procedures, a reduction in the degree of soil toxicity was achieved, with values ranging from 8.8 to 2.6 (Soil DW) and from 6.8 to 2.6 (Soil OS).
Soil toxicity at the consumer level was determined on the basis of the Ostracodtoxkit toxicological test, which showed that the use of phytoremediation supported by bioaugmentation with the B2 microbial consortium with γ-PGA caused the greatest decrease in the toxicity of soils polluted with petroleum hydrocarbons compared to Heterocypris incongruens. In the soil taken from the excavated pit area (Soil DW), the TU value decreased from 11.2 to 4.2 (Soil DW-5), and in the soil from the oil spill area (Soil OS), the TU value decreased from 10.9 to 3.6. The values of soil toxicity degrees after completion of phytoremediation supported by bioaugmentation with microbial consortium B1 with γ-PGA were 5.2 (Soil DW-4) and 4.2 (Soil OS-4). The highest toxicity values after remediation treatments were recorded in non-inoculated soils, at 10.2 (Soil DW-1) and 9.9 (Soil OS-1).
The use of Vibrio fischeri luminescent bacteria in the Microtox test made it possible to determine the toxicity of the tested soils at the level of decomposers. The values of the toxicity degrees of the initial Soil DW and Soil OS soils were 14.1 and 11.4, respectively. The remediation procedures carried out made it possible to reduce the toxicity levels of Soil DW to 12.3 (Soil DW-1), 8.5 (Soil DW-2), 6 (Soil DW-3), 5.4 (Soil DW-4), 4.4 (Soil DW-5) and 11.5 (Soil DW-6). In turn, the toxicity of Soil OS after 6 months of remediation treatments decreased to the levels of 10.5 (Soil OS-1), 7.8 (Soil OS-2), 5.4 (Soil OS-3), 4.6 (Soil OS -4), 3.9 (Soil OS-5) and 10.9 (Soil OS-6).
In addition, the MARA environmental risk assessment test was performed for each soil, which used 11 test strains with different sensitivity to xenobiotics present in the soil. The toxicities calculated based on the mean toxic concentration (MTC), expressed in toxicity units, were 14.3 (Soil DW) and 11.8 (Soil OS). As a result of the remediation treatments, the toxicity of soil DW decreased from 12.2 (in non-inoculated Soil DW-1) to 4.0 (in Soil DW-5). However, the values of Soil OS toxicity degrees after completion of remediation treatments ranged from 10.8 (Soil OS-1) to 3.4 (Soil OS-5).

4. Discussion

Phytoremediation is now a widely used method for remediating land contaminated with petroleum hydrocarbons, but for aged drilling waste or soils heavily contaminated with various types of xenobiotics, its effectiveness is usually too low. Therefore, so-called combined methods using micro-organisms are increasingly being used [18]. The study showed that the effectiveness of phytoremediation using Echinacea purpurea as a test plant largely depends on the concentration, type and age of the contaminant, as well as the conditions under which the treatment process is carried out [17]. The chromatographic analyses performed proved that the use of phytoremediation supported by inoculation with microbial consortia produced on the basis of indigenous micro-organisms allows for a significant reduction in the amount of TPHs and PAHs in the studied soils. This is probably due to the fact that the effectiveness of the soil remediation process using plants is affected by the number of bacteria, fungi and yeasts in the surrounding root zone [77]. Thus, the introduction of micro-organisms isolated from contaminated sites (biopreparation) into the soil makes it possible to improve the efficiency of contaminant elimination while avoiding antagonistic interactions of the soil’s indigenous microflora with foreign microbial cultures [9,78,79]. Moreover, inoculating the soil with the microbial consortia makes it possible to increase the concentration of micro-organisms near the roots, which influences their better growth [80]. The penetration of roots into the deeper layers of the rhizosphere causes the soil to expand, resulting in improved ventilation and permeability of the test soil, as well as increased interaction between micro-organisms and contamination. On the other hand, the plant’s roots secrete organic compounds, such as organic acids, sugars, amino acids, etc., which provide a source of carbon and energy for the growth of rhizosphere micro-organisms. It should also be remembered that dead root cells are treated by soil micro-organisms as an excellent source of nutrients, which further increases their metabolic activity [9,81,82,83]. Therefore, the synergistic interactions occurring among the micro-organisms present in the Echinacea purpurea root inoculant allow for an improvement in bioavailability to pollutants, thereby increasing their degradation efficiency.
Based on the chromatographic analyses of the soils, roots and shoots, it can be concluded that during phytoremediation studies using Echinacea purpurea, the petroleum-based contaminants underwent mechanisms of degradation, extraction, inhibition or a combination thereof [12,18]. Chromatographic analyses of the soils showed a slight reduction in the concentration of TPHs and PAHs in non-inoculated systems, while inoculation with microbial consortia and microbial consortia with γ-PGA allowed for a significant increase in the treatment efficiency of the studied soils. This proves the validity of the phytoremediation method assisted by microbial consortium inoculation. It should also be noted that among the inoculants tested, the B2 microbial consortium with γ-PGA was the most effective. This confirms the hypothesis that the use of an inoculant, which contains in its composition micro-organisms from different species (bacteria, fungi) and additives promoting the degradation of xenobiotics (γ-PGA), allows for an improvement in the efficiency of phytoremediation of soils, as well as biodegradation of oil pollutants by micro-organisms. Purification of the soils from toxic substances occurs due to the ability of the micro-organisms present in microbial consortia, natural soil microflora and Echinacea purpurea to produce enzymes and other chemicals, which break down or convert organic pollutants into simpler compounds with lower toxicity and better assimilation by plants [81,82]. Enzymes such as nitroreductase, dehalogenase, lactase, peroxidase and others are responsible for the degradation of petroleum hydrocarbons [84]. Moreover, the decrease in the concentration of petroleum hydrocarbons in the soil may be due to the transfer of contaminants from the soil to the plant [85,86,87]. This assumption is confirmed by the results of chromatographic analyses of the root and aboveground parts of Echinacea purpurea (stem + leaves). The experiments conducted showed the presence of petroleum hydrocarbons (TPHs and PAHs) in the roots and shoots of purple coneflower after the experiments. It is likely that during the treatment process of the studied soils, some of the analyzed contaminants were adsorbed on the root surface [20,88], and some were accumulated in the above-surface part of the plant via phytoextraction [18,87,89]. It should also be noted that some plants—for the proper conduct of the process of photosynthesis—use the ability to retain hydrocarbons in the tissues of the roots. This helps provide the energy needed for survival and is considered a self-defense strategy [87]. In addition, some contaminants can undergo phytoevaporation, which involves moving the contaminants into a volatile state [12,90]. It is also extremely important that γ-PGA was used as an additive to the microbial consortia in the study. Systems inoculated with γ-PGA-enhanced microbial consortia showed the highest % biodegradation of TPHs and PAHs, as well as the highest biomass production. γ-PGA provides an easily digestible source of carbon and energy for micro-organisms and Echinacea purpurea, which significantly affects the growth of the plant and the development of root zone micro-organisms, providing greater efficiency in the treatment of contaminated land.
Analyzing the effectiveness of the phytoremediation processes carried out in the systems studied, one cannot but mention the effect of the concentration and age of the contaminant on the efficiency of the treatment process. Remediation of aged soils (drilling waste) occurs much more slowly than soils contaminated by oil spills. This is related to the stronger adsorption of contaminants on the surface and in the porous space of aged soil [17,91]. Moreover, the study showed that the process of cleaning the soil from the tested contaminants proceeded at different rates depending on the type and dose of the toxicant. It was found that in soil containing a higher diversity of contaminants, the cleaning process was slower compared to soil from an oil spill site.
Toxicological tests carried out using bioindicators belonging to three taxonomic groups (bacteria, crustaceans, plants) confirmed the effectiveness of ongoing phytoremediation of soils contaminated with petroleum hydrocarbons in systems supported by inoculation with microbial consortia and microbial consortia with γ-PGA.

5. Conclusions

The purpose of this study was to evaluate the effectiveness of a phytoremediation method for soils contaminated with petroleum substances (TPHs and PAHs) supported by bioaugmentation with microbial consortia and microbial consortia with the addition of γ-PGA. For this study, we used Echinacea purpurea, characterized by an extensive fibrous root system, high tolerance for various soil conditions and environmental stresses, and proven ability to phytoremediate hydrocarbon-contaminated soils. The tests were conducted under laboratory conditions to ensure that the conditions were the same for all systems tested. On the basis of chromatographic analyses of the soils, roots and shoots, it was proved that combined methods (phytoremediation assisted by bioaugmentation) of remediation of land contaminated with petroleum hydrocarbons allow using the potential of both the process of biodegradation of contaminants by micro-organisms present in the inoculant and phytoremediation of land with the help of plant organisms. This makes it possible to significantly increase the efficiency of the remediation process of contaminated land and restore its usable properties, as confirmed by the results of the toxicological tests conducted.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app132413077/s1, Construction of B1 and B2 microbial consortia, Table S1: Identification of bacterial strains used in B1 and B2 microbial consortia.

Author Contributions

Conceptualization, K.W. and T.S.; software, K.W.; validation, K.W. and P.K.; formal analysis, K.W., T.S. and P.K.; investigation, K.W., T.S. and P.K.; writing—original draft preparation, K.W. and T.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Polish Ministry of Education and Science within statutory funding for Oil and Gas Institute—National Research Institute.

Institutional Review Board Statement

The studies used commercial toxicological tests not subject to ethical evaluation.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic of the stand for conducting bioaugmentation-assisted phytoremediation using Echinacea purpurea (laboratory conditions).
Figure 1. Schematic of the stand for conducting bioaugmentation-assisted phytoremediation using Echinacea purpurea (laboratory conditions).
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Figure 2. TPH content in soils after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems (System 1—soil DW + E. purpurea, System 2—soil DW + E. purpurea + B1 microbial consortium, System 3—soil DW + E. purpurea + B2 microbial consortium, System 4—soil DW + E. purpurea + B1 microbial consortium with the addition of γ-PGA, System 5—soil DW + E. purpurea + B2 microbial consortium with the addition of γ-PGA, System 6—soil DW + E. purpurea + γ-PGA, System 7—soil OS + E. purpurea, System 8—soil OS + E. purpurea + B1 microbial consortium, System 9—soil OS + E. purpurea + B2 microbial consortium, System 10—soil OS + E. purpurea + B1 microbial consortium with the addition of γ-PGA, System 11—soil OS + E. purpurea + B2 microbial consortium with the addition of γ-PGA, System 12—soil OS + E. purpurea + γ-PGA).
Figure 2. TPH content in soils after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems (System 1—soil DW + E. purpurea, System 2—soil DW + E. purpurea + B1 microbial consortium, System 3—soil DW + E. purpurea + B2 microbial consortium, System 4—soil DW + E. purpurea + B1 microbial consortium with the addition of γ-PGA, System 5—soil DW + E. purpurea + B2 microbial consortium with the addition of γ-PGA, System 6—soil DW + E. purpurea + γ-PGA, System 7—soil OS + E. purpurea, System 8—soil OS + E. purpurea + B1 microbial consortium, System 9—soil OS + E. purpurea + B2 microbial consortium, System 10—soil OS + E. purpurea + B1 microbial consortium with the addition of γ-PGA, System 11—soil OS + E. purpurea + B2 microbial consortium with the addition of γ-PGA, System 12—soil OS + E. purpurea + γ-PGA).
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Figure 3. TPH content of Echinacea purpurea roots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
Figure 3. TPH content of Echinacea purpurea roots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
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Figure 4. TPH content of Echinacea purpurea shoots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
Figure 4. TPH content of Echinacea purpurea shoots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
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Figure 5. PAH content in soil after 3 and 6 months of bioaugmentation-assisted phytoremediation process in the studied systems.
Figure 5. PAH content in soil after 3 and 6 months of bioaugmentation-assisted phytoremediation process in the studied systems.
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Figure 6. PAH content of Echinacea purpurea roots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
Figure 6. PAH content of Echinacea purpurea roots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
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Figure 7. PAH content of Echinacea purpurea shoots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
Figure 7. PAH content of Echinacea purpurea shoots after 3 and 6 months of phytoremediation process aided by bioaugmentation in the tested systems.
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Figure 8. Summary of the results of toxicological tests of drilling waste soil (Soil DW) subjected to the purification process in the analyzed systems (n = 3, p < 0.05).
Figure 8. Summary of the results of toxicological tests of drilling waste soil (Soil DW) subjected to the purification process in the analyzed systems (n = 3, p < 0.05).
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Figure 9. Summary of the results of toxicological tests of oil spill soil (Soil OS) subjected to the purification process in the analyzed systems (n = 3, p < 0.05).
Figure 9. Summary of the results of toxicological tests of oil spill soil (Soil OS) subjected to the purification process in the analyzed systems (n = 3, p < 0.05).
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Table 1. Results of physical and chemical analyses of soils used in studies of bioaugmentation-assisted phytoremediation process.
Table 1. Results of physical and chemical analyses of soils used in studies of bioaugmentation-assisted phytoremediation process.
ParameterSoil DWSoil OS
pH H2O7.066.99
Initial water moisture (%)21.923.01
BTEX (benzene, toluene, p-, m-ksylen)0.590.97
TPH5176.034025.65
WWA17.7110.30
Cl− a168.5258.6
S—SO42− a781.5826.3
N—NH4+ a70.179.4
N—NO3− a241.2230.4
P—PO43− a27.2632.1
Al2O3 b688.5426.9
SiO2 b236.5315.6
Fe2O3 b5.36.2
MgO b3.93.0
CaO b6.96.5
Sand (%)54.0175.21
Silt (%)10.376.92
Clay (%)35.6217.87
Heavy metal content aAs0.90.7
Ba28.923.5
Cd1.00.7
Cr11.19.1
Co3.13.0
Cu22.224.5
Hg0.50.2
Pb22.915.4
Mo1.82.1
Sn5.46.7
Zn20.617.1
Ni11.89.3
COD—Chemical oxygen demand, a Content expressed as ((mg kg−1 dry mass), b Content expressed as (g kg−1 dry mass).
Table 2. Operating parameters of chromatographs during the determination of TPHs and PAHs.
Table 2. Operating parameters of chromatographs during the determination of TPHs and PAHs.
Chromatographic Analysis of Aliphatic Hydrocarbons—TPH, nC6–nC44, Pristane, Phytane (Clarus 500 GC Perkin Elmer)
Parameter of Chromatography
Temperature (°C)ColumnDetectorCalibration Standards
InjectorCarrier GasTemperature Program
290 °CHe
20 mL min−1		30 °C—isothermal run for 2 min
30–105 °C—temp. increase rate 10 °C min−1
105–285 °C—temp. increase rate 5 °C min−1
285 °C—isothermal run for 5 min−1		RTX-1
30 m × 0.53 mm (Restek, Bellefonte, PA, USA)		FID
300 °C		Reference soil: BAM—K010 (Tusnovic Instruments, Poland)
Standard mixture hydrocarbons (nC6–nC44) ASTM®
No. D2807 (Supelco, Saint Louis, MO, USA)
Fuel Oil Degradation Mix nC17, pristane, nC18, phytane No. A029668 (Restek, Bellefonte, PA, USA)
Chromatographic Analysis of Polycyclic Aromatic Hydrocarbons—PAH (Vanquish Core Thermo Scientific)
Parameter of Chromatography
EluentsFlowGradientColumnDetectorCalibration Standards
A—methanol
B—acetonitrile
(Chempur, Piekary śląskie, Poland)		1.5 mL min−1 20% B—for 1.5 min
20–50% B—for 1.5 min
50–100% B—for 1 min
100% B—for 1 min
100–0% B—for 3 min
100% A—for 3 min		NUCLEODUR C18 PAH column
125 mm × 4 mm, 3 µm (Marcherey-Nagel, Dueren, Germany)		UV-ViS
FLD		Reference soil: PAHs by HPLC No. SQC017-40G (Sigma-Aldrich, Saint Louis, MO, USA)
Certified PAH-Mix solution No. 722393 (Marcherey-Nagel, Dueren, Germany)
Table 3. Contents of selected groups of aliphatic hydrocarbons in the studied soils (Soil DW and Soil OS) before and after treatment with phytoremediation assisted by inoculation.
Table 3. Contents of selected groups of aliphatic hydrocarbons in the studied soils (Soil DW and Soil OS) before and after treatment with phytoremediation assisted by inoculation.
ParameterContent ± SD (mg/kg Dry Mass Soil)
Initial Soil DWAfter 180 Days
Soil DW-1Soil DW-2Soil DW-3Soil DW-4Soil DW-5Soil DW-6
TPH5176.034223.453335.332890.12758.112381.964047.68
∑nC6–nC926.3120.367.583.442.521.9818.75
∑nC10–nC21892.48683.89407.46300.37271.65196.37627.57
∑nC22–nC301092.38919.85845.72845.72777.35661.4896.15
∑nC31–nC36383.94347.88333.86316.11317.99290.3342.39
isoprenoids 169.93159.37147.65142.73141.63135.23156.52
Unidentified aliphatic hydrocarbons2610.982092.11593.071347.241246.981096.692006.31
ParameterInitial Soil OSAfter 180 Days
Soil OS-1Soil OS-2Soil OS-3Soil OS-4Soil OS-5Soil OS-6
TPH4025.653145.082381.172021.521965.031591.262943.24
∑nC6–nC94.092.990.80.460.390.282.75
∑nC10–nC211013.77735.64422.35352.87258.55191.87658.24
∑nC22–nC30358.69290.79263.5263.5231.71193.99285.91
∑nC31–nC36200.59179.49172.95153.23164.76144.65176.54
isoprenoids384.97356.53325.83319.22309.93300.32348.62
Unidentified aliphatic hydrocarbons2063.541579.641195.74963.57999.69760.151471.18
Table 4. Contents of selected groups of PAHs in the studied soils (Soil DW and Soil OS) before and after treatment with phytoremediation assisted by inoculation.
Table 4. Contents of selected groups of PAHs in the studied soils (Soil DW and Soil OS) before and after treatment with phytoremediation assisted by inoculation.
ParameterContent ± SD (mg/kg Dry Mass Soil)
Initial Soil DWAfter 180 Days
Soil DW-1Soil DW-2Soil DW-3Soil DW-4Soil DW-5Soil DW-6
∑PAH17.7114.8512.2511.20410.768.9514.19
∑two-ring PAHs5.9854.6673.3092.7952.64624.361
∑ three-ring PAHs4.8153.9613.2162.8132.8251.6393.763
∑ four-ring PAHs5.1964.6554.2634.163.9183.5024.529
∑ five-ring PAHs1.2781.1681.0871.0651.0170.9521.143
∑ six-ring PAHs0.4340.40.3770.370.3580.340.392
ParameterInitial Soil OSAfter 180 Days
Soil OS-1Soil OS-2Soil OS-3Soil OS-4Soil OS-5Soil OS-6
∑PAHs10.38.6267.517.16.926.348.43
∑two-ring PAHs0.4950.3350.1950.1530.150.1290.316
∑ three-ring PAHs1.3250.9790.7280.6120.6390.3810.938
∑ four-ring PAHs5.0264.2753.8083.7043.5082.4684.187
∑ five-ring PAHs2.3222.031.851.7361.7271.6511.995
∑ six-ring PAHs1.1361.0080.930.8980.8910.8590.993
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Wojtowicz, K.; Steliga, T.; Kapusta, P. Evaluation of the Effectiveness of Bioaugmentation-Assisted Phytoremediation of Soils Contaminated with Petroleum Hydrocarbons Using Echinacea purpurea. Appl. Sci. 2023, 13, 13077. https://doi.org/10.3390/app132413077

AMA Style

Wojtowicz K, Steliga T, Kapusta P. Evaluation of the Effectiveness of Bioaugmentation-Assisted Phytoremediation of Soils Contaminated with Petroleum Hydrocarbons Using Echinacea purpurea. Applied Sciences. 2023; 13(24):13077. https://doi.org/10.3390/app132413077

Chicago/Turabian Style

Wojtowicz, Katarzyna, Teresa Steliga, and Piotr Kapusta. 2023. "Evaluation of the Effectiveness of Bioaugmentation-Assisted Phytoremediation of Soils Contaminated with Petroleum Hydrocarbons Using Echinacea purpurea" Applied Sciences 13, no. 24: 13077. https://doi.org/10.3390/app132413077

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