NL2027994B1 - Process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride jx993849 - Google Patents
Process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride jx993849 Download PDFInfo
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Abstract
The invention is related to the ?eld of soil remediation technologies, and in particular to a process for remediation of heavy metal-containing soil by using pig manure and 5 lrichoderma virl'de J X993 849, comprising steps of mixing pig manure With soil to be treated at a mixing ratio of l to 5 % by mass to cultivate a plant, and watering the soil near roots of the plant with a fermentation broth from lrichoderma viride J X993 849. The process enables the pH level of moderately alkaline soil to be reduced and active heavy metal content in the soil to be increased, and can promote the absorption of the 10 heavy metal by the plant, thereby achieving remediation of the soil.
Description
PROCESS FOR REMEDIATION OF HEAVY METAL-CONTAINING SOIL
BY USING PIG MANURE AND TRICHODERMA VIRIDE JX993849
The present invention is related to the field of soil remediation technologies, and in particular to a process for remediation of heavy metal-containing soil by using pig manure and frichoderma viride JX993849.
Saline-alkali soil is an important reserve land resource. However, its exploitation and production security are seriously threatened in recent years due to the heavy metal (such as, cadmium (Cd), lead (Pd), and mercury (Hg)) contamination.
Saline-alkali soil has a poor soil structure and buffering power due to the high pH value. This may cause adverse effects on plant growth and availability of heavy metals.
There are currently many studies done on how to remove heavy metals from soil.
Among these, phytoremediation, that uses plants to clean up pollution in soil, is an economic and effective solution. However, activities of the plants used may be inhibited and even lost due to the presence of certain metals or other materials in soil, or absorption of heavy metals by the plants in different areas of the target soil may vary greatly due to the different heavy metal concentrations in the areas. Therefore, the current phytoremediation technologies cannot achieve a reduction in the heavy metal contents in soil in an efficient manner.
In view of the above problems, an objective of the invention is to provide a process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride JX993849, which can enable the pH level of moderately alkaline soil to be reduced and active heavy metal contents in soil to be increased, and can promote the absorption of heavy metals by plants.
Accordingly, an objective of the invention is realized by a process for remediation of heavy metal-containing soil by using pig manure and frichoderma viride JX993849, comprising:
mixing pig manure with soil to be treated at a mixing ratio of 1 to 5 % by mass to cultivate a plant, and watering the soil near roots of the plant with a fermentation broth from trichoderma viride JX993849.
In a preferred embodiment, the fermentation broth has a quantity of spores ranging from 5x108 to 8.5%10° spores/ml.
In a preferred embodiment, the soil near roots of the plant is watered two times with the fermentation broth, with the first watering being performed at day 15 after emergence of the plant and the second being performed at day 35 after emergence of the plant. The amount of the fermentation broth used each time may be 10 to 20 % by mass with respect to the mass of the soil to be treated.
In a preferred embodiment, the plant comprises a plant which tends to accumulate a heavy metal.
In a further preferred embodiment, the plant, which tends to accumulate a heavy metal, comprises Artemisia scoparia.
Ina preferred embodiment, the soil remediation comprises reduction of the pH level of moderately alkaline soil, and increasement of the active heavy metal content in soil and enhanced absorption of the heavy metal by the plant.
In a preferred embodiment, the heavy metal comprises one or more of mercury (Hg), cadmium (Cd), and lead (Pb).
The process of the present invention has several advantages. The invention provides a process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride JX993849, comprising mixing pig manure with soil to be treated at a mixing ratio of 1 to 5 % by mass to cultivate a plant, and watering the soil near roots of the plant with a fermentation broth from trichoderma viride JX993849. The process can enable the pH level of moderately alkaline soil to be reduced and the active heavy metal content in soil to be increased, and can promote the absorption of the heavy metal by the plant. Experiments, as described hereinafter, have shown that, with the process of the invention, the pH value of the moderately alkaline soil sample to be treated was decreased by 0.65 to 0.74; the contents of active heavy metals in the soil were increased; and the absorption of the heavy metals by the plant was promoted.
Further, the experiments have also shown that, with the process of the invention, the biomasses of aerial and subterranean parts of the Artemisia scoparia plant were increased by 7.3 to 62.3 % and 56.8 to 198.8 %, respectively, and the absorption of the heavy metals by the Artemisia scoparia palnt was promoted.
Name and Address of Depository Institution at Which the Biological Materials
Were Deposited
Trichoderma virens JX993849 was deposited under the accession number CGMCC
No. 3.17613 in January, 2015 at China General Microbiological Culture Collection
Center (CGMCC).
Unless otherwise noted, all starting materials used herein are commercially available.
The invention provides a process for remediation of heavy metal-containing soil by using pig manure and trichoderma viride JX993849, comprising: mixing pig manure with soil to be treated at a mixing ratio of 1 to 5 % by mass to cultivate a plant, and watering the soil near roots of the plant with a fermentation broth from trichoderma viride JX993849.
According to the process, pig manure is mixed with soil to be treated so as to cultivate a plant, and the soil near roots of the plant is watered with a fermentation broth from trichoderma viride JX993849. The pig manure is used at an amount of 1 to 5 %, preferably 1.5 to 4.5 %, with respect to the mass of the soil to be treated. There is no specific limitation concerning the origin of the pig manure. In an embodiment, the pig manure is originated from natural fermentation of pig farm waste. The fermentation broth from frichoderma viride JX993849 preferably has a quantity of spores ranging from 5x10% to 8.5x10° spores/ml, more preferably 7.5x108 to 1x10” spores/ml.
According to the process of the invention, the pig manure serves to supply plant nutrients and thus to improve the soil. The fermentation broth serves to reduce the pH value of the soil, and promote growth of the plant and absorption of the heavy metal by the plant. The process of the invention can enable the pH value of moderately alkaline soil to be reduced and the active heavy metal content in the soil to be increased, and thus promote the absorption of the heavy metal by the plant, thereby achieving remediation of the soil.
In this description, the soil sample selected for treatment through the process of the invention had a total Hg content of 23.5 mg/kg, a total Pd content of 45.2 mg/kg, a total Cd content of 1.08 mg/kg, a cation exchange capacity of 19.8 cmol/kg, an organic content of 16.6 g/kg, and a pH value of 8.62.
In the examples described hereinafter, flowerpots, having a diameter of 40 cm and a height of 75 cm, were used for performing the investigation on soil remediation through the process of the invention, with each pot containing 800 g of the soil. The mixing and watering are not particularly limited herein and may be performed in any manner well known to a skilled person in the art.
The watering is preferably performed two times. Preferably, the first watering is performed at day 15 after emergence of the plant, and the second watering is performed at day 35 after emergence of the plant. The amount of the fermentation broth used each time is preferably 10 to 20 % by mass with respect to the mass of the soil to be treated. Further preferably, the amounts of the fermentation broth used by the first and second watering are 10 % and 15 %, respectively, with respect to the mass of the soil to be treated. The plant, as described above, preferably comprises a plant which tends to accumulate a heavy metal. The plant, which tends to accumulate a heavy metal, preferably comprises Artemisia scoparia. Species of the plants of
Artemisia scoparia is not particularly limited herein, and commercially available one may be employed.
The preparation of the fermentation broth from trichoderma viride JX993849 is not particularly limited, and may be performed in any manner well known to a skilled person in the art. A preferred preparation method comprises: inoculating trichoderma viride JX993849 into potato dextrose agar (PDA) solid medium (potato (peeled): 200 g, dextrose: 20 g, water: 1000 ml, natural pH) for activation to obtain activated trichoderma viride 1X993849; inoculating the activated trichoderma viride JX993849 into PDA slant medium (potato (peeled): 200 g, dextrose: 20 g, agar: 20 g, water: 1000 ml, natural pH) for conidia culture to obtain a conidia suspension; and inoculating the conidia suspension into PDA liquid medium for fermentation to obtain the fermentation broth from trichoderma viride JX993849.
The activation is preferably carried out at 28 °C for 36 hours. The conidia culture is preferably carried out at 28 °C for 6 days. The fermentation is preferably carried out at 28 to 30 °C for 60 hours with an agitation speed of 180 to 200 rpm. The fermentation broth from frichoderma viride JX993849 can adjust the pH value of the soil, produce auxin (IAA), dissolve phosphorus, and secrete siderophore.
The soil remediation preferably comprises reduction of the pH level of moderately alkaline soil, increasement of the active heavy metal content in soil, and enhanced absorption of the heavy metal by the plant. The heavy metal preferably comprises one or more of Hg, Cd, and Pb.
The process of the invention can enable the pH level of moderately alkaline soil to be reduced and the biomasses of aerial and subterranean parts of the Artemisia scoparia plant to be increased, and can also promote the absorption of the heavy metal by the Artemisia scoparia plant. In particular, experiments, as described hereinafter, have shown that, with the process of the invention, the pH value of the moderately alkaline soil sample was decreased by 0.65 to 0.74; the biomasses of aerial and subterranean parts of the Artemisia scoparia plant were increased by 7.3 to 62.3 % and 56.8 to 198.8 %, respectively; the accumulated amounts of Cd, Pd, and Hg in the aerial part of the Artemisia scoparia plant were increased from 0.65, 3.62, and 20.6 mg/kg to 1.57, 9.64, and 50.4 mg/kg, respectively; and the accumulated amounts of Cd, Pd, and Hg in the subterranean part of the Artemisia scoparia plant were increased from 1.51, 7.61, and 33.3 mg/kg to 4.87, 19.3, and 63.2 mg/kg.
In order to further illustrate the invention, the process for remediation of heavy metal- containing soil by using pig manure and trichoderma viride JX993849 according to the invention will be further described with respect to the following examples, which, however, are not intended to limit the scope of the invention.
Example 1
A fermentation broth from trichoderma viride JX993849 was prepared by the following steps.
Trichoderma viride JX993849 was inoculated into PDA solid medium for activation to obtain activated trichoderma viride JX993849,
The activated trichoderma viride JX993849 was inoculated into PDA slant medium for conidia culture. The conidia culture was carried out at 28 °C for 6 d to obtain conidia after being rinsed using sterile water. Dilution was then performed so as to obtain a conidia suspension having a spore concentration of 1x10’ spores/ml.
The conidia suspension was inoculated under aseptic conditions into PDA liquid medium, and then incubated at 28 to 30 °C for 60 h with a stirring speed of 180 to 200 rpm to obtain a fermentation broth from trichoderma viride JX993849, which had a quantity of spores ranging from 5x10% to 1x10’ spores/ml.
Application Example 1
The example was performed as an experiment in the Chemistry Building at Qingdao
Agricultural University, conducted from October, 2017 to April, 2018. A soil sample used had physicochemical properties as shown in Table 1.
Table 1 Soil physicochemical properties © Organic Cation exchange capacity Total Cd Total Pd Total Hg pH matter/(g/kg) /(cmol/kg) mg/kg) /(mg/kg) /(mg/kg) 862 166 198 108 452 235
Flowerpots were divided into 6 experimental groups (3 replicates per group). Various groups are shown in table 2.
Table 2 Amounts of pig manure and fermentation broth from trichoderma viride
JX993849 used in various groups
Fermentation broth from trichoderma viride
Group Pig manure JX993849 /(% by mass) /(% by mass) tT 0 0 2 1 0 3 5 0 4 0 25 5 1 25 6 5 25
In particular, pig manure was well mixed with the soil sample. 800 g of the mixture was then placed into each flowerpot with an inner diameter of 40 cm and a height of 75 cm. Thereafter, Artemisia scoparia seeds were planted in the soil contained in each pot. During growth of the plants, water content in the soil contained in each pot was maintained at around 65 % of the maximum field capacity.
Atday 15 after emergence of the Artemisia scoparia plants, the soil near roots of the plant in each pot was watered with the fermentation broth in an amount of 10 % with respect to the mass of the soil. At day 35 after emergence of the Artemisia scoparia plants, the soil near roots of the plant in each pot was then watered again with the fermentation broth in an amount of 15 % with respect to the mass of the soil.
After 70 days, all plants were harvested and the soil in each pot was sampled, for later experiments.
<7-
Application Example 2
The soil, in Application Example 1, contained in each pot after harvest of the plants was subjected to determination of pH. The results are shown in Table 3.
Table 3 Soil pH values
Group PH
Ta 2 7.452 3 7.23a 4 7.56b 7.2b 6 6.82b 5 Note: different letters denote significant differences between the treatments (at p<0.05).
Table 3 shows that there was a significant difference between the groups subjected to the fermentation broth and the groups not subjected to the fermentation broth, and the pH of the soil subjected to the fermentation broth was decreased. In particular, as compared with Group 1, the pH of the soil in Group 4 was decreased by 0.23; as compared with Group 2, the pH of the soil in Group 5 was decreased by 0.25; and as compared with Group 3, the pH of the soil in Group 6 was decreased by 0.41.
Therefore, with the process of the invention, the pH of the soil was decreased.
Application Example 3
Forms of Hg, Cd, and Pd present in the soil samples obtained in Application Example 1 were determined by a BCR sequential extraction method. The results are shown in
Table 4.
Table 4 Heavy metal forms in soil
© Forms of heavy metalinsoil
Heavy me metal Group Acid- Reducible Oxidizable Residual extractable 1 18% 30% 23% 30% 2 20% 36% 26% 18% 3 29% 37% 18% 17% ca 4 30% 36% 16% 18% 37% 37% 21% 5% 6 48% 27% 20% 5% 5% ATA 2 35% 36% 13% 17% 3 32% 42% 9% 17% pd 4 38% 34% 20% 8% 5 38% 32% 20% 10% 6 49% 36% 12% 3% 1 16% 23% 21% 41% 2 24% 23% 21% 32% 3 26% 24% 21% 29%
He 4 27% 28% 23% 22% 5 36% 30% 25% 9% 6 38% 31% 27% 4%
Table 4 shows that, the content of the metal Cd in a residual form present in the soil samples in Group 2 was decreased as compared with Group 1 and the content thereof in Group 3 was further decreased as compared with Group 2. The content of the metal
Cd in an acid-extractable form present in the soil samples in Group 2 was increased as 5 compared with Group 1 and the content thereof in Group 3 was further increased as compared with Group 2. In particular, the content of Cd in the acid-extractable form was increased from 18 % in Group 1 to 29 % in Group 3. Further, the contents of Cd in the residual, oxidizable, and reducible forms present in the soil samples in Groups 2 and 3 were decreased as compared with Group 1. The content of Cd in the residual form was decreased from 30 % in Group 1 to 17 % in Group 3, and in Group 3, the content of Cd in the residual form was the lowest among all the forms. These indicate that the reducible and oxidizable forms were mutually converted into the other.
Further, it can also be seen from Table 4 that, in Groups 4, 5, and 6, the reducible, oxidizable, and residual forms were gradually converted into the acid-extractable form,
which can be readily taken up and utilized by the plants, and especially, in Group 6, the content of Cd in the acid-extractable form reached up to 48 % with respect to the total amount of the metal Cd. Therefore, the process of the invention promoted conversion of the residual and reducible forms of Cd into the acid-extractable form thereof, thereby improving bio-availability of the metal Cd.
With respect to Hg, it can be seen that, in Groups 1 to 3, as the added amount of pig manure was increased, the content of Hg in the residual form present in the soil samples decreased from 41 % in Group 1 to 29 % in Group 3 and the content of Hg in the acid-extractable form increased from 14 % to 26 %. In groups 4 to 6, the forms were the acid-extractable form, the oxidizable form, the reducible form, and the residual form when arranged from the highest. This indicates that the residual, reducible, and oxidizable forms were all gradually converted into the acid-extractable form as the addition of pig manure. Further, the content of Hg in the acid-extractable form present in the soil samples in Groups 4 to 6 was higher than that of Hg in the other forms, and the content thereof was increased from 27 % in Group 4 to 38 % in
Group 6. Therefore, the content of Hg in the acid-extractable form in the soil was substantially improved through the process of the invention.
With respect to Pd, it can be seen from Table 4 that, as compared with Group 1, the contents of Pd in the residual form present in the soil samples in Groups 2 and 3 were decreased, and the contents of Pd in the acid-extractable form were increased, in particularly increased from 25 % in Group 1 to 35 % in Group 2. Further, the contents of Pd in the residual, oxidizable, and reducible forms present in the soil samples in
Groups 2 and 3 were decreased as compared with Group 1. In particular, the content of
Pd in the residual form was decreased from 24 % in Group 1 to 17 % in Group 3, and the content of Pd in the residual form was the lowest among all the forms. These indicate that the reducible and oxidizable forms of the metal Pd were mutually converted into the other. Further, it can also be seen from Table 4 that, in Groups 4 to 6, the reducible, oxidizable, and residual forms were gradually converted into the acid- extractable form, which can be readily taken up and utilized by the plants; and especially, in Group 6, the content of Pd in the acid-extractable form reached up to 49 % with respect to the total amount of the metal Pd. Therefore, the process of the invention enabled the bio-availability of Pd to be improved.
Application Example 4
Biomass of the plants harvested in Application Example 1 were determined. The results are shown in Table 5.
Table 5 Biomass of the Artemisia scoparia plants
Group Aerialpart OO Subterraneanpart /(g/pot) /(g/pot) 1 0.212b 0.542b 2 0.332b 0.580b 3 0.415b 0.655b 4 0.243a 0.624a 0.462a 0.798a 6 0.634a 0.877a
Table 5 shows that, as compared with Group 1, the biomass of the subterranean part in 5 Groups 2 and 3 were increased by 56.8 % and 95.8 %, respectively; and the biomass of the subterranean part in Groups 5 and 6 were increased by 117.7 % and 198.8 %, respectively. Further, as compared with Group 1, the biomass of the aerial part in
Groups 2 and 3 were increased by 7.3 % and 21.2 %, respectively; and the biomass of the aerial part in Groups 5 and 6 were increased by 47.7 % and 62.3 %, respectively.
Among the six groups, the plants in Group 6 grew best. These indicate that the use of trichoderma viride alone or pig manure alone promoted growth of the plants, but trichoderma viride used in combination with pig manure produced significantly better effects than any one of trichoderma viride and pig manure. Therefore, the process of the invention enabled the biomass of the Artemisia scoparia plants to be significantly improved.
Application Example 5
The heavy metal contents in the Artemisia scoparia plants harvested in Application
Example 1 were measured. In particular, the Cd and Pd contents were measured by an
ICP-MS method, and the Hg content was measured through Atomic Fluorescence
Spectrometry (AFS). The results are shown in Tables 6 to 8.
Table 6 Cd contents in the Artemisia scoparia plants
Heawmetal Group AcrialPart Sublerrancan Part [(mg/kg) [(mg/kg) 1 065m 151b
Cd 2 0.832b 2.33b 3 1.40b 4.35b
Aerial Part Subterranean Part
Heavy metal Group (mg/kg) mg/kg) 4 0.941a 2.54a 1.05a 3.15a 6 157a 4.87a
Table 7 Pd contents in the Artemisia scoparia plants
Aerial Part Subterranean Part
Heavy metal Group (mg/kg) mg/kg) 1 3.62b 7.61b 2 4.28b 8.98b 3 5.76b 12.1b
Pd 4 5.37a 10.7a 5 7.26a 14.5a 6 9.64b 193a
Table 8 Hg contents in the Artemisia scoparia plants
Aerial Part Subterranean Part
Heavy metal Group
I(mg/kg) I(mg/kg) 1 20.6b 33.3b 2 27 9b 44 8b 3 31.4b 56.6b
Hg 4 35.4a 37.24 5 40.5a 47.02 6 50.4a 63.2a
Tables 6 to 8 show that there was a significant difference between the groups subjected to trichoderma viride and the groups not subjected to #richoderma viride with both 5 subjected to the same treatment of pig manure. In particular, as compared with Group 1, the Cd contents in the subterranean parts in Groups 2 and 3 were increased by 54.3 % and 188 %, respectively, and the Cd contents in the subterranean parts in
Groups 5 and 6 were increased by 108 % and 223 %, respectively. Further, the Cd content in the subterranean part in Group 6 was the highest. Further, as compared with
Group 1, the Cd contents in the aerial parts in Groups 2 and 3 were increased by 26.7 % and 113 %, respectively; and the Cd contents in the aerial parts in Groups 5 and 6 were increased by 59.7 % and 139 %, respectively. Therefore, absorption of the metal Cd by the Artemisia scoparia plants was promoted by the process of the invention, and thus the Cd-contaminated soil was efficiently improved.
With respect to Hg, as compared with Group 1, the Hg contents in the subterranean parts in Groups 2 and 3 were increased by 34.5 % and 69.9 %, respectively; and the Hg contents in the subterranean parts in Groups 5 and 6 were increased by 41.2 % and 89.9 %, respectively. Further, as compared with Group 1, the Hg contents in the aerial parts in Groups 2 and 3 were increased by 35.4 % and 52.4 %, respectively; and the Hg contents in the aerial parts in Groups 5 and 6 were increased by 96.7 % and 144 %, respectively. These indicate that pig manure used in combination with trichoderma viride significantly promoted absorption of the metal Hg by the plants. Therefore, absorption of Hg by the Artemisia scoparia plants was promoted by the process of the invention, and thus the Hg-contaminated soil was efficiently improved.
With respect to Pd, as compared with Group 1, the Pd contents in the subterranean parts in Groups 2 and 3 were increased by 18.0 % and 59.0 %, respectively; and the Pd contents in the subterranean parts in Groups 5 and 6 were increased by 90.8 % and 153.3 %, respectively. Further, as compared with Group 1, the Pd contents in the aerial parts in Groups 2 and 3 were increased by 18.2 % and 59.2 %, respectively, and the Pd contents in the aerial parts in Groups 5 and 6 were increased by 101 % and 166 %, respectively. These indicate that the use of trichoderma viride alone or pig manure alone promoted growth of the plants, but frichoderma viride used in combination with pig manure produced significantly better effects than any one of #richoderma viride and pig manure. Therefore, absorption of Pd by the Artemisia scoparia plants was improved by the process of the invention, and the Pd-contaminated soil was efficiently improved.
While preferred embodiments of the present invention have been described, it should be understood that the present invention is not so limited and various alterations and modifications may be made without departing from the scope and spirit of the present invention. The scope of the invention is to be limited only by the appended claims.
Claims (7)
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