Method for strengthening phytoremediation of heavy metal cadmium contaminated soil

Abstract

The invention is applicable to the technical field of soil remediation, and particularly relates to a method for enhancing phytoremediation of heavy metal cadmium contaminated soil, which comprises the following steps: planting hyperaccumulators in to-be-remediated heavy metal cadmium contaminated soil, and regularly applying bacterial liquid to the soil at the roots of the hyperaccumulators in the growth cycle of the hyperaccumulators. According to the method, inoculation of the thiobacillus ferrooxidans in the soil has no influence on the soil environment, growth of hyperaccumulators and growth, yield and quality of crops, the price is low, and large-area popularization and application can be achieved in practical application.

Description

Technical Field

[0001] The invention belongs to the technical field of soil remediation, and in particular relates to a method for strengthening phytoremediation of soil contaminated by heavy metal cadmium. Background Art

[0002] Cadmium is considered to be the most toxic heavy metal to human health. It can damage human kidney function, make bones fragile, cause osteoporosis, and increase human carcinogenicity, causing kidney cancer, bladder cancer, breast cancer and prostate cancer. Therefore, it is imperative to strengthen the restoration and management of soil contaminated by heavy metal cadmium to ensure the safe production of agricultural products and human health.

[0003] It is worth noting that most of the microbial agents used in the prior art increase the effective Cd content in the soil by secreting organic acids, amino acids and biosurfactants, thereby improving the remediation efficiency of plants. Although it can effectively promote the absorption and accumulation of soil heavy metal Cd by hyperaccumulators, it also has problems such as soil acidification and other physiological and biochemical damage to the soil. Summary of the invention

[0004] The purpose of the present invention is to provide a method for strengthening phytoremediation of heavy metal cadmium contaminated soil. Although the existing technology can effectively promote the absorption and accumulation of soil heavy metal Cd by hyperaccumulators, it also has the problem of causing soil physiological and biochemical damage such as soil acidification.

[0005] The present invention is achieved by providing a method for strengthening plant repair of heavy metal cadmium contaminated soil, the method comprising: planting hyperaccumulator plants in the heavy metal cadmium contaminated soil to be repaired, and regularly applying bacterial liquid to the root soil of the hyperaccumulator plants during the growth cycle of the hyperaccumulator plants.

[0006] Preferably, the bacterial solution applied to the root soil of the hyperaccumulator plant is Thiobacillus ferrooxidans, which is rod-shaped and a Gram-negative bacterium.

[0007] Preferably, in the step of applying the bacterial solution to the soil at the roots of the hyperaccumulator plants, the depth of application of the bacterial solution is 0.2-0.6 cm from the soil surface.

[0008] Preferably, the concentration of the bacterial solution is 4.5×10 7 ~1.0×10 8 CFU / mL; the volume of each inoculation is 1.5-2.5mL.

[0009] Preferably, the frequency of applying the bacterial solution to the root soil of the hyper-enriched plants is once every 6-9 days, for a total of 3-4 times.

[0010] Preferably, the hyperaccumulator plants are at least one or more of cosmos, nightshade, sedum austriatum, centipede grass, shepherd's purse, elsholtzia ciliata and pokeweed.

[0011] Preferably, when the hyperaccumulator plant is cosmos, cosmos with two leaves and a root length of 2.0 to 2.5 cm is transplanted into the heavy metal cadmium contaminated soil to be remediated.

[0012] Preferably, after the cosmos is transplanted to the heavy metal cadmium contaminated soil to be repaired, the bacterial solution is inoculated after waiting for 1 to 2 weeks.

[0013] Preferably, the bacterial solution is applied to a depth of 0.3 cm from the soil surface.

[0014] Preferably, the volume of each inoculation is 2 mL.

[0015] The method for strengthening phytoremediation of heavy metal cadmium contaminated soil provided by the present invention has the following beneficial effects:

[0016] Compared with the hyperaccumulator plant cosmos not inoculated with Thiobacillus ferrooxidans to repair the soil, the Cd content in the hyperaccumulator plant cosmos planted on the heavy metal Cd contaminated soil can be increased by more than 51%, thereby improving the efficiency of hyperaccumulator plants in repairing the heavy metal Cd contaminated soil.

[0017] The invention inoculates Thiobacillus ferrooxidans in the soil, has no influence on the soil environment, the growth of hyperaccumulator plants and the growth, yield and quality of crops, has a low price and can be widely promoted and applied in practical applications. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] An embodiment of the present invention provides a method for enhanced phytoremediation of heavy metal cadmium contaminated soil, the method comprising: planting hyperaccumulator plants in the heavy metal cadmium contaminated soil to be remediated, and regularly applying bacterial solution to the root soil of the hyperaccumulator plants during the growth cycle of the hyperaccumulator plants.

[0020] In an embodiment of the present invention, the repair hyperaccumulator plant is planted in the heavy metal Cd contaminated soil to be repaired, and during the growth cycle of the hyperaccumulator plant, the soil at the root of the hyperaccumulator plant is regularly inoculated with a bacterial solution of Thiobacillus ferrooxidans. The Thiobacillus ferrooxidans is isolated from the waste residue of a certain abandoned pyrite mine in Hunan Province and is an acidophilic Thiobacillus ferrooxidans.

[0021] The bacterial solution is inoculated to a depth of 0.2 to 0.5 cm on the soil surface where the roots of hyperaccumulators are repaired. More preferably, it is 0.3 cm. This thickness is suitable for practical operation and can effectively activate the soil heavy metal Cd near the developed root system of hyperaccumulators, which is beneficial to the absorption and accumulation of effective Cd in the soil by the developed root system of hyperaccumulators.

[0022] The concentration of the bacterial solution is 4.5×107~1.0×108CFU / mL; the volume of the bacterial solution for each inoculation is 1.5~2.5mL. Further preferably, the volume of the bacterial solution for each inoculation is 2.0mL. This inoculation dose can enable the strain to have the best colonization effect. Inoculate once every 6 to 9 days, for a total of 3 to 4 times. The best case is to inoculate once a week, for a total of 4 times. This inoculation time and number of times can enable the number of live bacteria of Thiobacillus ferrooxidans in the heavy metal Cd contaminated soil to continue to maintain a high level, so that Thiobacillus ferrooxidans can better activate the heavy metal Cd in the soil and increase the effective Cd content in the soil.

[0023] The present invention uses the super-accumulator plant cosmos which has a strong accumulation ability of heavy metal Cd in soil as the test plant. The repair super-accumulator plants applicable to the method of the present invention include but are not limited to super-accumulator plants of heavy metal Cd such as Solanum nigrum, Sedum austriatum, Scolopendra subspinipes, Shepherd's purse, Elsholtzia marissima and Phytolacca americana.

[0024] When using the hyperaccumulator cosmos as a hyperaccumulator, after the plant grows to two leaves and a root length of 1.8 to 2.2 cm, it is transplanted into the farmland to be repaired that is contaminated with heavy metal Cd; after the plant is cultivated in the farmland to be repaired for 7 to 14 days, the bacterial solution is inoculated. If the hyperaccumulator cosmos is inoculated too early after transplantation, it is not conducive to the recovery growth of the hyperaccumulator cosmos in the soil contaminated with heavy metal Cd; if the hyperaccumulator cosmos is inoculated too late after transplantation, the accumulation of heavy metal Cd in the hyperaccumulator cosmos by the inoculated Thiobacillus ferrooxidans will be weakened, affecting its repair effect.

[0025] The Cd content in the heavy metal cadmium contaminated soil is 2.0mg / kg to 6.0mg / kg. When the heavy metal Cd content in the soil is lower than 2.0mg / kg or higher than 6.0mg / kg,

[0026] The Ni content in the heavy metal Cd-contaminated soil is 40 mg / kg to 200 mg / kg. If the Ni content in the soil is lower than 40 mg / kg or higher than 200 mg / kg, the promoting effect on restoration after inoculation is reduced by about 10 to 30%.

[0027] In the following experiments, cosmos, which has a strong ability to accumulate heavy metal Cd, was used as the test plant.

[0028] Embodiment 1:

[0029] The test plant in this experiment is the Cd hyperaccumulator plant cosmos. The soil was collected from a place in Hunan Province that was contaminated with heavy metal Cd. The Cd content was 2.78±0.36mg / kg and the pH value was 5.26. The soil was naturally air-dried after being taken back to the laboratory. After the soil was mixed, it was passed through a 5mm sieve and sterilized by high pressure (121℃, 30min) before use. A potted plant experiment was carried out using a perforated plastic pot (150×105×115mm) with a plastic tray. Each plastic pot was filled with 200g of air-dried soil samples for use, and 3 replicates were used. Ammonium bicarbonate, superphosphate and potassium sulfate were used as basal fertilizers, applied at a ratio of 0.112, 0.045 and 0.067g / kg, respectively, and water was added for balance for 10d after stirring evenly.

[0030] After the seeds of cosmos were treated in 2% H2O2 for 15 min, they were washed three times with deionized water and sown in vermiculite for seedling cultivation. When the cosmos seedlings grew to two leaves and the roots were about 2 cm long, three cosmos seedlings of the same size were transplanted into each culture pot. The plants were cultured in an artificial climate chamber under the following conditions: light intensity of 50-60 μmol photons / m 2 ·s, relative humidity 70% to 80%, photoperiod 12h / 25℃ / day, 12h / 22℃ / night. In order to keep the soil moisture content at 60% to 70%, water was added every 2 to 3 days in this example.

[0031] After culturing the cosmos in a culture pot for 2 weeks, the ferrothiobacillus was inoculated. Methods of cultivating and treating the bacterial agent: the above-mentioned strains were inoculated into 9K culture medium for enrichment culture. The culture medium formula (1L) was (NH4)2SO43.0g, KCl 0.1g, Ca(NO3)2·4H2O 0.01g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 44.3g, distilled water 1000mL, pH 2.0, sterilized at 121℃ for 30min. The inoculated strain bottle was placed in a constant temperature shaking box for 3 days (speed 150r / min, temperature 28℃). The activated bacterial solution was placed at 4000r / min for 5min, the supernatant was discarded, 10mL of 0.9% saline was added, vortexed and mixed, and then centrifuged and washed twice. Inoculate 2 mL of the above bacterial solution at a depth of 0.5 cm in the soil surface layer at the root of each cosmos. Apply once a week for a total of 3 times. Set up a blank control group without inoculating Thiobacillus ferrooxidans.

[0032] The cosmos were harvested after being cultured in an artificial climate chamber for 45 days, rinsed with tap water, and then rinsed three times with distilled water, and then dried in an oven at 75°C to constant weight. The fresh weight and dry weight were recorded separately. After drying, the samples were ground for testing.

[0033] Weigh the dried and ground sample (0.25 g) in a digestion tube, add 8 mL HNO3 and soak overnight, then digest in a DigiBlock ED54 digestion system, treat at 80 ° C for 1.5 h, 120 ° C for 1.5 h, 150 ° C for 3 h, and 170 ° C for acid removal (0.5-1 h). When 1-2 mL of digestion solution remains in the digestion tube, the digestion is terminated; then deionized water is added to make the volume 50 mL, and after filtering, the heavy metal Cd content in the digestion solution is determined by ICP-MS. The measured values ​​are calibrated using spinach standard material (SRM 1570a, National Institute of Standards and Technology, NIST), and duplicate and blank samples are added to each batch of samples for quality control.

[0034] The heavy metal Cd content in the upper stems and leaves of peacock grass (by dry weight, the same below) is shown in Table 1, and the Cd content in the soil is shown in Table 2:

[0035] Table 1 Cd content and accumulation in the aboveground parts of soil contaminated with heavy metal Cd after planting cosmos

[0036]

[0037]

[0038] Table 2. Soil Cd removal under heavy metal Cd contaminated soil remediation

[0039]

[0040] Example 2

[0041] The test plant in this experiment is the Cd hyperaccumulator plant cosmos. The soil was collected from a place in Hunan Province that was contaminated with heavy metal Cd. The Cd content was 3.27±0.43 mg / kg and the pH value was 5.62. The soil was naturally air-dried after being taken back to the laboratory. After the soil was mixed, it was passed through a 5mm sieve and sterilized by high pressure (121℃, 30min) for use. A potted plant experiment was carried out using a perforated plastic pot (150×105×115mm) with a plastic tray. Each plastic pot was filled with 200g of air-dried soil samples for use, and 3 replicates were used. Ammonium bicarbonate, superphosphate and potassium sulfate were used as basal fertilizers, applied at a ratio of 0.112, 0.045 and 0.067g / kg, respectively, and water was added for balance for 10d after stirring evenly.

[0042] After the seeds of cosmos were treated in 2% H2O2 for 15 min, they were washed three times with deionized water and sown in vermiculite for seedling cultivation. When the cosmos seedlings grew to two leaves and the roots were about 2 cm long, three cosmos seedlings of the same size were transplanted into each culture pot. The plants were cultured in an artificial climate chamber under the following conditions: light intensity of 50-60 μmol photons / m 2 ·s, relative humidity 70% to 80%, photoperiod 12h / 25℃ / day, 12h / 22℃ / night. In order to keep the soil moisture content at 60% to 70%, water was added every 2 to 3 days in this example.

[0043] After culturing the cosmos in a culture pot for 2 weeks, inoculate with Thiobacillus ferrooxidans. Inoculate 2 mL of the bacterial solution at a depth of 0.5 cm in the soil surface layer of each cosmos root. Apply once a week for a total of 3 times. Set up a blank control group without inoculating with Thiobacillus ferrooxidans.

[0044] The cosmos were harvested after being cultured in an artificial climate chamber for 45 days, rinsed with tap water, and then rinsed three times with distilled water, and then dried in an oven at 75°C to constant weight. The fresh weight and dry weight were recorded separately. After drying, the samples were ground for testing.

[0045] Weigh the dried and ground sample (0.25g) in a digestion tube, add 8mL HNO3 and soak overnight, then digest in DigiBlock ED54 digestion system, treat at 80℃ for 1.5h, 120℃ for 1.5h, 150℃ for 3h, and 170℃ for acid removal (0.5-1h). When 1-2mL digestion solution is left in the digestion tube, stop digestion; then add deionized water to 50mL, filter, and use ICP-MS to determine the heavy metal Cd content in the digestion solution. Use spinach standard material (SRM 1570a, National Institute of Standards and Technology, NIST) to calibrate the measured value, and add duplicate and blank samples to each batch of samples for quality control. The experimental results are shown in Tables 3 and 4 below.

[0046] The heavy metal Cd content in the upper stems and leaves of peacock grass (calculated by dry weight, the same below) is shown in Table 3, and the Cd content in the soil is shown in Table 4:

[0047] Table 3 Cd content and accumulation in the aboveground parts of soil contaminated with heavy metal Cd after planting cosmos

[0048]

[0049] Table 4. Soil Cd removal under heavy metal Cd contaminated soil remediation

[0050]

[0051] Example 3

[0052] The plant used in this experiment is Solanum nigrum, a superaccumulator of heavy metal Cd. The soil was collected from a place in Hunan Province that was contaminated with heavy metal Cd. The Cd content was 2.78±0.36mg / kg and the pH value was 5.26. The soil was naturally air-dried after being taken back to the laboratory. After the soil was mixed, it was passed through a 5mm sieve and sterilized by high pressure (121℃, 30min) for use. The potted plant experiment was carried out in a perforated plastic pot (150×105×115mm) with a plastic tray. Each plastic pot was filled with 200g of air-dried soil sample for use, and 3 replicates were used. Ammonium bicarbonate, superphosphate and potassium sulfate were used as basal fertilizers, applied at a ratio of 0.112, 0.045 and 0.067g / kg, respectively, and water was added for balance for 10d after stirring evenly.

[0053] Solanum nigrum seeds were treated in 2% H2O2 for 15 min, washed three times with deionized water, and sown in vermiculite for seedling cultivation. When the cosmos seedlings grew to two leaves and the roots were about 2 cm long, three Solanum nigrum seedlings of the same size were transplanted into each culture pot. The plants were cultured in an artificial climate chamber under the following conditions: light intensity 50-60 μmol photons / m 2·s, relative humidity 70% to 80%, photoperiod 12h / 25℃ / day, 12h / 22℃ / night. In order to keep the soil moisture content at 60% to 70%, water was added every 2 to 3 days in this example.

[0054] After the nightshade was cultured in a culture pot for 2 weeks, it was inoculated with Thiobacillus ferrooxidans. 2 mL of the above bacterial solution was inoculated at a depth of 0.5 cm in the soil surface layer of each nightshade root. The solution was applied once a week for a total of 3 times. A blank control group was set up without inoculation of Thiobacillus ferrooxidans.

[0055] Solanum nigrum was harvested after 45 days of cultivation in an artificial climate chamber, rinsed with tap water, and then rinsed three times with distilled water, inactivated and dried in an oven at 75°C to constant weight, and the fresh weight and dry weight were recorded respectively. After drying, the samples were ground for testing.

[0056] Weigh the dried and ground sample (0.25g) in a digestion tube, add 8mL HNO3 and soak overnight, then digest in a DigiBlock ED54 digestion system, treat at 80℃ for 1.5h, 120℃ for 1.5h, 150℃ for 3h, and 170℃ for acid removal (0.5-1h). When 1-2mL of digestion solution is left in the digestion tube, digestion is terminated; then deionized water is added to make the volume 50mL, filtered, and the heavy metal Cd content in the digestion solution is determined by ICP-MS. Spinach standard material (SRM1570a, National Institute of Standards and Technology, NIST) is used to calibrate the measured value, and duplicate and blank samples are added to each batch of samples for quality control. The experimental results are shown in Tables 5 and 6 below.

[0057] The heavy metal Cd content in the aboveground stems and leaves of Solanum nigrum (calculated by dry weight, the same below) is shown in Table 5, and the Cd content in the soil is shown in Table 6:

[0058] Table 5 Cd content and accumulation in the aboveground parts of Solanum nigrum under the remediation of heavy metal Cd contaminated soil

[0059]

[0060] Table 6 Soil Cd removal under heavy metal Cd contaminated soil remediation

[0061]

[0062] Example 4

[0063] The plant used in this experiment was Solanum nigrum, a superaccumulator of heavy metal Cd. The soil was collected from a place in Hunan Province that was contaminated with heavy metal Cd. The Cd content was 3.27±0.43 mg / kg and the pH value was 5.62. The soil was naturally air-dried after being taken back to the laboratory. After the soil was mixed, it was passed through a 5 mm sieve and sterilized by high pressure (121°C, 30 min) for use. A potted plant experiment was carried out using a perforated plastic pot (150×105×115 mm) with a plastic tray. Each plastic pot was filled with 200 g of air-dried soil samples for use, and 3 replicates were used. Ammonium bicarbonate, superphosphate and potassium sulfate were used as basal fertilizers, applied at a ratio of 0.112, 0.045 and 0.067 g / kg, respectively, and water was added for balance for 10 days after stirring evenly.

[0064] Solanum nigrum seeds were treated in 2% H2O2 for 15 min, washed three times with deionized water, and sown in vermiculite for seedling cultivation. When the cosmos seedlings grew to two leaves and the roots were about 2 cm long, three Solanum nigrum seedlings of the same size were transplanted into each culture pot. The plants were cultured in an artificial climate chamber under the following conditions: light intensity 50-60 μmol photons / m 2 ·s, relative humidity 70% to 80%, photoperiod 12h / 25℃ / day, 12h / 22℃ / night. In order to keep the soil moisture content at 60% to 70%, water was added every 2 to 3 days in this example.

[0065] After the nightshade was cultured in a culture pot for 2 weeks, it was inoculated with Thiobacillus ferrooxidans. 2 mL of the above bacterial solution was inoculated at a depth of 0.5 cm in the soil surface layer of each nightshade root. The solution was applied once a week for a total of 3 times. A blank control group was set up without inoculation of Thiobacillus ferrooxidans.

[0066] Solanum nigrum was harvested after 45 days of cultivation in an artificial climate chamber, rinsed with tap water, and then rinsed three times with distilled water, inactivated and dried in an oven at 75°C to constant weight, and the fresh weight and dry weight were recorded respectively. After drying, the samples were ground for testing.

[0067] Weigh the dried and ground sample (0.25g) in a digestion tube, add 8mL HNO3 and soak overnight, then digest in a DigiBlock ED54 digestion system, treat at 80 degrees Celsius for 1.5h, 120 degrees Celsius for 1.5h, 150 degrees Celsius for 3h, and 170 degrees Celsius for acid removal (0.5-1h). When 1-2mL of digestion solution remains in the digestion tube, the digestion is terminated; then deionized water is added to make the volume 50mL, and after filtering, the heavy metal Cd content in the digestion solution is determined by ICP-MS. The measured values ​​are calibrated using spinach standard material (SRM1570a, National Institute of Standards and Technology, NIST), and duplicate and blank samples are added to each batch of samples for quality control. The experimental results are shown in Tables 7 and 8 below.

[0068] The heavy metal Cd content in the aboveground stems and leaves of Solanum nigrum (calculated by dry weight, the same below) is shown in Table 7, and the Cd content in the soil is shown in Table 8:

[0069] Table 7 Cd content and accumulation in the aboveground parts of Solanum nigrum under the restoration of heavy metal Cd contaminated soil

[0070]

[0071] Table 8 Soil Cd removal under heavy metal Cd contaminated soil remediation

[0072]

[0073] It can be seen from the experimental data that the heavy metal Cd contaminated soil inoculation with Thiobacillus ferrooxidans to enhance super-enrichment phytoremediation technology of the present invention is feasible for the remediation of Cd-contaminated soil with heavy metals, and the effect is good.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for strengthening phytoremediation of heavy metal cadmium contaminated soil, characterized in that: The method comprises: planting the hyperaccumulator plant in the heavy metal cadmium contaminated soil to be repaired, and regularly applying bacterial liquid to the root soil of the hyperaccumulator plant during the growth cycle of the hyperaccumulator plant.

2. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 1, characterized in that: The bacterial solution applied to the root soil of the hyperaccumulator plants is Thiobacillus ferrooxidans, which is a rod-shaped, Gram-negative bacterium.

3. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 1, characterized in that: In the step of applying the bacterial solution to the soil at the roots of the hyperaccumulator plants, the depth of application of the bacterial solution is 0.2-0.6 cm from the soil surface.

4. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 1, characterized in that: The concentration of the bacterial solution was 4.5×10 7 ~1.0×10 8 CFU / mL; the volume of each inoculation is 1.5-2.5mL.

5. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 1, characterized in that: The frequency of applying the bacterial solution to the root soil of the hyperaccumulator plants is once every 6-9 days, for a total of 3-4 times.

6. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 1, characterized in that: The hyperaccumulator plants are at least one or more of cosmos, nightshade, sedum austriatum, centipede grass, shepherd's purse, elsholtzia ciliata and pokeweed.

7. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 6, characterized in that: When the hyperaccumulator plant is cosmos, cosmos with two leaves and a root length of 2.0 to 2.5 cm is transplanted into the heavy metal cadmium contaminated soil to be remediated.

8. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 7, characterized in that: After transplanting the cosmos to the heavy metal cadmium-contaminated soil to be repaired, wait for 1 to 2 weeks before inoculating the bacterial solution.

9. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 3, characterized in that: The depth of bacterial solution application was 0.3 cm from the soil surface.

10. The method for strengthening phytoremediation of heavy metal cadmium contaminated soil according to claim 4, characterized in that: The volume of each inoculation was 2 mL.

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