CN114643275B - Method for in-situ remediation of arsenic in soil by bacteria and application - Google Patents
Method for in-situ remediation of arsenic in soil by bacteria and application Download PDFInfo
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- CN114643275B CN114643275B CN202210169420.7A CN202210169420A CN114643275B CN 114643275 B CN114643275 B CN 114643275B CN 202210169420 A CN202210169420 A CN 202210169420A CN 114643275 B CN114643275 B CN 114643275B
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- 239000002689 soil Substances 0.000 title claims abstract description 80
- 229910052785 arsenic Inorganic materials 0.000 title claims abstract description 78
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 30
- 241000894006 Bacteria Species 0.000 title claims abstract description 17
- 238000005067 remediation Methods 0.000 title claims abstract description 17
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 11
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 claims abstract description 90
- 239000011572 manganese Substances 0.000 claims abstract description 27
- 239000000463 material Substances 0.000 claims abstract description 24
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910052748 manganese Inorganic materials 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 18
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 14
- 230000008439 repair process Effects 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 241000589291 Acinetobacter Species 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 229940099596 manganese sulfate Drugs 0.000 claims description 8
- 239000011702 manganese sulphate Substances 0.000 claims description 8
- 235000007079 manganese sulphate Nutrition 0.000 claims description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 7
- 238000009631 Broth culture Methods 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 239000001099 ammonium carbonate Substances 0.000 claims description 7
- 235000012501 ammonium carbonate Nutrition 0.000 claims description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims description 7
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 229960004642 ferric ammonium citrate Drugs 0.000 claims description 7
- 239000004313 iron ammonium citrate Substances 0.000 claims description 7
- 235000000011 iron ammonium citrate Nutrition 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 235000010344 sodium nitrate Nutrition 0.000 claims description 7
- 239000004317 sodium nitrate Substances 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 241000736262 Microbiota Species 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 230000007613 environmental effect Effects 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 238000005842 biochemical reaction Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 13
- 238000002386 leaching Methods 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 6
- 230000006378 damage Effects 0.000 description 4
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 4
- 238000007711 solidification Methods 0.000 description 4
- 230000008023 solidification Effects 0.000 description 4
- 230000009471 action Effects 0.000 description 3
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- 239000011435 rock Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000008316 Arsenic Poisoning Diseases 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101000794816 Pseudomonas putida Anthranilate synthase component 1 Proteins 0.000 description 1
- 101000847784 Pseudomonas putida Anthranilate synthase component 2 Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- LULLIKNODDLMDQ-UHFFFAOYSA-N arsenic(3+) Chemical compound [As+3] LULLIKNODDLMDQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 238000003895 groundwater pollution Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
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- 210000003734 kidney Anatomy 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 229910052752 metalloid Inorganic materials 0.000 description 1
- 150000002738 metalloids Chemical class 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
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- 150000007522 mineralic acids Chemical class 0.000 description 1
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- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
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- 150000007524 organic acids Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229910052683 pyrite Inorganic materials 0.000 description 1
- NIFIFKQPDTWWGU-UHFFFAOYSA-N pyrite Chemical compound [Fe+2].[S-][S-] NIFIFKQPDTWWGU-UHFFFAOYSA-N 0.000 description 1
- 239000011028 pyrite Substances 0.000 description 1
- 229910052957 realgar Inorganic materials 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/08—Reclamation of contaminated soil chemically
Abstract
The invention discloses a method for in-situ remediation of arsenic in soil by bacteria and application thereof, comprising the following steps: after enrichment culture, the strain is added into arsenic-polluted soil, after components such as carbon source and divalent manganese are added, the water content of the soil is regulated to be 20-40%, the soil is stirred for 10-30min by a stirrer, and then the strain is placed under room temperature for 2-3 weeks, so that biological manganese oxide materials induced by bacteria and trivalent arsenic or pentavalent arsenic in the soil undergo physical-chemical-biochemical reaction, and unstable arsenic in the soil can be converted into stable arsenic, thereby achieving the aim of restoring the arsenic in the soil. The method is simple to operate, high in adjustability and suitable for repairing large-area arsenic-polluted soil.
Description
Technical Field
The invention relates to the technical field of soil treatment, in particular to a method for in-situ remediation of arsenic in soil by bacteria and application thereof.
Background
Arsenic is widely present in natural environments and is a toxic metalloid. In natural environment, arsenic exists in crust, igneous rock and sedimentary rock, mainly in the form of pyrite, realgar, estrus arsenic and other sulphide minerals. Human activities such as the use of chemical fertilizers, pesticides, and mining activities, promote arsenic accumulation in groundwater and farmlands. Arsenic is classified as a dangerous substance by the U.S. poison and disease register because of its severe toxicity, and the dangerous level is higher than that of toxic metals such as cadmium, lead, mercury, etc. The long-term environment with high arsenic can cause serious injury to human health. The human being can contact arsenic through drinking water and food chains, and arsenic can accumulate in a large amount in liver and kidney organs of the human body. Related pathological studies have found that if drinking water or food with excessive arsenic content is taken for a long period of time, arsenic poisoning can be caused, shock, renal failure can be seriously caused, and even death occurs due to multi-organ failure. Long-term exposure to high arsenic environments may cause genetic mutations, greatly increasing the risk of cancer.
The prior art on which arsenic-contaminated soil treatment depends mainly includes soil leaching, solidification/stabilization, heat treatment, phytoremediation, microorganism remediation and other technologies. The soil leaching is usually carried out by adding various leaching agents (such as organic or inorganic acid, alkali, salt and chelating agent) into the soil, and the method can damage the original micro-aggregate structure of the soil, can cause leaching loss and precipitation of nutrient elements, is easy to cause groundwater pollution, and is only suitable for the soil treatment with small area and heavy pollution. The heat treatment of arsenic-contaminated soil also damages the soil structure and also faces the problems of high treatment cost and running cost. The solidification/stabilization technology has many applications in the treatment of arsenic contaminated soil engineering, such as cement encapsulation solidification, chelant stabilization, metal oxide and other inorganic materials adsorption solidification, but the dosage of general medicaments is usually more than 5%, the primary cost is high, and secondary pollution is easy to generate. The phytoremediation method has the advantages of low cost, no damage to the original structure of the soil and small change of physicochemical properties of the soil, but has lower remediation efficiency and long remediation period. As an emerging repair technology, microbial repair has the advantages of good effect, economy and no secondary pollution, has proved to be an effective repair method, and is concerned by researchers at home and abroad.
Disclosure of Invention
The invention aims to provide a method for in-situ remediation of arsenic in soil by bacteria, so as to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for in-situ remediation of arsenic in soil by bacteria, S11: adding bacterial liquid capable of inducing to produce biological manganese oxide material into arsenic-polluted soil, and sequentially adding a carbon source and a divalent manganese component;
s12: regulating the water content of the soil to 20-40%, stirring for 10-30min by a stirrer, and then culturing for 2-3 weeks at room temperature to enable the biological manganese oxide material to have physical-chemical reaction with trivalent arsenic or pentavalent arsenic in the soil so as to finish the repair of the arsenic in the soil;
preferably, the strain is a strain that converts divalent manganese to tetravalent manganese oxide.
Preferably, the strain is specifically Acinetobacter bieleisii WHW-2.
Preferably, the preparation steps of the biological manganese oxide material specifically include: the strain is inoculated in 17-25g/L nutrient broth culture medium for enrichment culture.
Preferably, the conditions of the enrichment culture are: aerobic enrichment culture is carried out for 3-5 days at 15-30 ℃ under horizontal oscillation with frequency of 100-180 rpm; in the enrichment culture process, the transfer quantity of the strain is 2-10% according to the volume ratio.
The enriched strain is added into 50g arsenic polluted soil, and after bacterial liquid is added, 16mg peptone, 4mg yeast extract powder, 2mg potassium dihydrogen phosphate, 4mg magnesium sulfate, 4mg sodium nitrate, 2mg calcium chloride, 2mg ammonium chloride, 20mg ferric ammonium citrate, 4mg manganese sulfate and 2mg ammonium carbonate are sequentially added.
Preferably, the bacterial is added in an amount of 0.8-1.5mL per gram of soil in terms of liquid-solid ratio.
Preferably, the water content of the soil is adjusted to 25%, stirred by a stirrer for 25min, and then the soil is cultured for 3 weeks at room temperature.
On the other hand, the invention also provides application of the method for in-situ remediation of arsenic in soil by the bacteria in environmental remediation engineering.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the method, bacteria are not required to be loaded on a carrier, the operation is simple, the adjustability is high, divalent manganese is closely contacted with polluted soil in a manganese oxide mode through the action of a bacterial strain, the arsenic restoration effect is effectively improved, arsenic in an exchangeable state in the soil can be converted into arsenic in a residue state under the action of a bacterial strain metabolite by using the method for restoring arsenic polluted soil by using biological manganese oxide, so that the mobility and biological effectiveness of the arsenic in the soil are reduced, and the arsenic in the soil is effectively fixed.
2. The invention has the advantages of simple process, short repair time, convenient operation, low treatment cost, large treatment range, no secondary pollution and the like; the biological manganese oxide material is added into arsenic polluted soil along with the bacterial liquid, so that a series of physical-chemical reactions are carried out on the biological manganese oxide material and trivalent arsenic or pentavalent arsenic in the soil, and the mobility of arsenic in the soil, particularly trivalent arsenic, is reduced, so that the aim of arsenic remediation is fulfilled.
Drawings
FIG. 1 shows the OD in example 1 of the present invention 600 A change in manganese oxide content;
FIG. 2 shows the content changes of divalent Mn and tetravalent Mn in example 1 of the present invention;
FIG. 3 is a lineage development tree of the strain used in the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As described in the background art, no biological manganese oxide is used for repairing arsenic polluted soil in the prior art, the method can reduce the migration and biological effectiveness of arsenic in the soil to a certain extent, and the effect is remarkable.
In order to achieve the above object, the first aspect of the present invention provides a method for culturing bacteria, comprising the steps of separating and purifying the strain, specifically:
(1) Separation and domestication of bacterial strains:
firstly, adding a water sample with mixed microbiota into a nutrient broth culture medium, culturing for 72 hours, transferring bacterial liquid in the nutrient broth culture medium into a PYCM culture medium (0.8 g/L peptone, 0.2g/L yeast extract, 0.1g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L sodium nitrate, 0.1g/L calcium chloride, 0.1g/L ammonium chloride, 1g/L ferric ammonium citrate, 0.2g/L manganese sulfate and 0.1g/L ammonium carbonate), and continuously domesticating.
Step-by-step dilution: the bacterial liquid in PYCM is absorbed according to 10 -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 ，10 -6 ，10 -7 ，10 -8 Is diluted in a gradient.
Coating: firstly pouring the flat plate, solidifying the flat plate, then pouring the bacterial liquid onto the flat plate, rapidly coating the surface of the flat plate back and forth by using a coater, uniformly distributing the bacterial liquid, and then growing uniform single bacterial colonies.
Plate scribing: preparing a PYCM solid plate, picking a single colony by an inoculating loop, streaking the single colony onto the solid plate, repeating the streaking for a plurality of times to obtain a pure colony, and finally identifying and naming the acinetobacter bauhinii WHW-2 by pedigree identification.
Inoculating the obtained strain into nutrient broth for enrichment culture;
inoculating the strain after enrichment culture into a culture medium containing bivalent manganese for culture, and generally obtaining the manganese oxide material induced by bacteria after 2-3 days of culture.
The strain is a strain capable of converting divalent manganese into manganese oxide, and the strain is specifically Acinetobacter bieleisii WHW-2.
The invention introduces bacterial liquid after 5 days of culture into a soil system, and the divalent manganese in the culture medium basically reaches a stable level after 5 days of culture.
The biological method of strain culture is adopted to convert the divalent manganese into the manganese oxide material, rather than simply preparing the manganese oxide material by chemical oxidation, because the biological method has the following advantages compared with the conventional chemical reaction process: (1) The reaction condition is milder, the environment is similar to neutral, a large amount of acid, alkaline reagent and toxic and harmful reagent are avoided, and the whole preparation process is more environment-friendly; (2) Compared with inorganic manganese (IV) oxide, the biological manganese (IV) oxide has higher specific surface area and higher adsorption efficiency on arsenic;
in one or more embodiments of the present invention, a method for preparing a biological manganese oxide material specifically includes the steps of:
1) Inoculating the strain into a culture medium for enrichment culture;
2) Preparing a culture medium containing divalent manganese;
3) Adding the enriched strain into the culture medium, carrying out shaking culture under certain conditions, and centrifuging at a high speed after 5 days to obtain the biological manganese oxide material.
In one or more embodiments of the invention, the strain is Acinetobacter biers WHW-2.
Specifically, the strain enrichment medium is a medium capable of playing a role in strain enrichment in the field, and the components of the medium can be adaptively adjusted for different types of strains; taking pseudomonas putida as an example, as a preferred embodiment, the composition of the culture medium is: nutrient broth (19 g/l).
Further, the conditions of enrichment culture are: the strain grows well under the condition of aerobic enrichment culture for 1-5 days at 15-35 ℃ and horizontal shaking (100-180 rpm).
Further, in the enrichment culture process, the transfer amount of the strain is 2-10%.
In one or more embodiments of the present invention, the medium containing divalent manganese is any medium capable of maintaining normal metabolism of a strain to which divalent manganese is added, and as a preferred example, the preparation method of the medium specifically includes: a culture medium was prepared by adding 300mL of the culture medium to a 500mL conical flask, and adjusting pH=7.+ -. 0.2, wherein the culture medium comprises 0.8g/L of peptone, 0.2g/L of yeast extract, 0.1g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate, 0.2g/L of sodium nitrate, 0.1g/L of calcium chloride, 0.1g/L of ammonium chloride, 1.0g/L of ferric ammonium citrate, 0.2g/L of manganese sulfate and 0.1g/L of ammonium carbonate.
Further, to avoid other strainsAffecting the activity of the strain to be inoculated, avoiding other strains from generating byproducts by metabolism, and sterilizing the strain after the preparation of the divalent manganese culture medium is finished, wherein the sterilization treatment time is 30-40min, preferably 30min. The conditions of shaking culture were: placing in shaking incubator at 30-35deg.C and 180r.min -1 Shaking culture was carried out under the condition for 5 days.
The effect of bivalent manganese and bacterial strain is different and affects each other, so the addition amount has an important effect on the effect of repairing the arsenic polluted soil. Wherein the manganese oxide converted from divalent manganese has oxidizing ability to arsenic, trivalent arsenic is adsorbed on the surface of manganese oxide and oxidized into pentavalent arsenic, and the pentavalent arsenic is subjected to coordination reaction on the surface of manganese oxide to form As (V) -MnO 2 Bidentate dinuclear bridged complexes. When the addition amount of the divalent manganese is too high, the whole process is mainly oxidized, the adsorption capacity is reduced, arsenic cannot be enriched on the surface of manganese oxide, and the high-efficiency restoration of the arsenic-containing soil is not utilized; thus, as a preferred example, divalent manganese is added in the form of manganese sulfate, the manganese being added in an amount of 65mg/L;
the second aspect of the invention provides a method for in-situ remediation of arsenic in soil by bacteria, S11: adding bacterial liquid capable of inducing to produce biological manganese oxide material into arsenic-polluted soil, and sequentially adding a carbon source and a divalent manganese component;
s12: regulating the water content of the soil to 20-40%, stirring for 10-30min by a stirrer, and then culturing for 2-3 weeks at room temperature to enable the biological manganese oxide material to have physical-chemical reaction with trivalent arsenic or pentavalent arsenic in the soil so as to finish the repair of the arsenic in the soil;
further, the strain adopts a strain for converting divalent manganese into tetravalent manganese oxide, and the strain is specifically Acinetobacter bieleisii WHW-2.
Specifically, the preparation steps of the biological manganese oxide material specifically include: the strain is inoculated in 17-25g/L nutrient broth culture medium for enrichment culture.
It is worth noting that the conditions for enrichment culture are: aerobic enrichment culture is carried out for 3-5 days at 15-30 ℃ under horizontal oscillation with frequency of 100-180 rpm; in the enrichment culture process, the transfer quantity of the strain is 2-10% according to the volume ratio.
Further, the enriched strain is added into 50g arsenic contaminated soil, and after bacterial liquid is added, 16mg peptone, 4mg yeast extract powder, 2mg monopotassium phosphate, 4mg magnesium sulfate, 4mg sodium nitrate, 2mg calcium chloride, 2mg ammonium chloride, 20mg ferric ammonium citrate, 4mg manganese sulfate and 2mg ammonium carbonate are sequentially added.
It is notable that the addition amount of bacteria is 0.8-1.5mL per gram of soil according to the liquid-solid ratio.
In addition, the water content of the soil was adjusted to 25%, stirred by a stirrer for 25min, and then left to stand at room temperature for 3 weeks.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
Enrichment culture of bacterial strains:
the bacterial strain is Acinetobacter bieleisii WHW-2. Inoculating the bacteria into enrichment culture medium (19 g/l nutrient broth) according to 2-10% of transfer amount by volume ratio, and performing aerobic enrichment culture for 1-5 days under horizontal shaking (100-180 rpm) at 15-35 ℃;
as can be seen from FIG. 1, after 5 days of culture, the culture was performed at OD 600 The concentration increasing trend of the represented biological manganese oxide tends to be gentle, and the biological manganese oxide can be used for repairing arsenic-polluted soil at the moment.
As can be seen from FIG. 2, after 5 days of culture, the concentration of Mn (II) in the culture medium is already lower than 4mg/L, the Mn (II) continues to be converted into Mn (IV) oxide slowly, and the biological Mn oxide can be used for repairing arsenic-polluted soil.
Example 2
Preparation of biological manganese oxide:
1) Preparation of the culture medium: peptone 0.8g/L, yeast extract 0.2g/L, dipotassium hydrogen phosphate 0.1g/L, magnesium sulfate 0.2g/L, sodium nitrate 0.2g/L, calcium chloride 0.1g/L, ammonium chloride 0.1g/L, ferric ammonium citrate 1.0g/L, manganese sulfate 0.2g/L, and ammonium carbonate 0.1g/L, were prepared to prepare a medium, and 300mL of the medium was added to a 500mL conical flask to adjust pH=7.+ -. 0.2. Sterilizing for 30min after the preparation is completed.
The strain was added to the sterilized medium. Placing it in a shaking incubator at 150 r.min -1 Shaking culture at 30℃for 5 days. After the oscillation is completed, the solid powder is obtained by centrifugal suction filtration, namely the biological manganese oxide material.
2) The repairing process comprises the following steps: adding the biological manganese oxide material prepared in the step (2) into arsenic-polluted soil for repairing;
the biological manganese oxide material prepared in the step (1) is added into 50g of arsenic-polluted soil according to the proportion of 1 percent (0.5 g), the water content of the soil is regulated to 40 percent, the mixture is stirred for 20 minutes by a stirrer, and then the mixture is placed under the room temperature condition (25 ℃) for 2 weeks, and the experiment is terminated.
As can be seen from the data in the table, the arsenic content of the embodiment is reduced by water leaching and TCLP leaching, the arsenic content is reduced from 0.14mg/L to 0.09mg/L when not repaired after water leaching, and the repair efficiency is 35.71%; the TCLP is reduced from unrepaired 0.18mg/L to 0.08mg/L, and the repair efficiency is 55.56%.
Example 3
On the basis of example 2, arsenic-contaminated soil was continuously adjusted to have a water content of 40%, stirred with a stirrer for 20 minutes, left to stand at room temperature (25 ℃) and after 2 weeks of incubation, the restoration effect was analyzed.
As can be seen from the data in the table, the arsenic content of the embodiment is reduced, the arsenic content is reduced from 0.14mg/L to 0.08mg/L when not repaired after water leaching, and the repair efficiency is 42.86%; the TCLP is reduced from unrepaired 0.18mg/L to 0.07mg/L, and the repair efficiency is 61.11%.
And (3) effect analysis:
as can be seen from the above embodiments: the arsenic content of the biological manganese oxide material for treating the arsenic-containing soil is obviously reduced, which indicates that the material can achieve an excellent effect of repairing the arsenic-polluted soil.
The repairing process comprises the following steps:
trivalent arsenic has higher activity, toxicity and migration capability than pentavalent arsenic, and has stronger adhesion capability and mobility, so that the removal of trivalent arsenic is considered in the repair work of arsenic-polluted soil.
The manganese oxide is stable at normal temperature, has a loose structure, has high surface charge, large specific surface area and rich surface hydroxyl groups-OH, has double functions of oxidization and adsorption, and can be used as a better stabilizing and repairing material for the soil polluted by the arsenic and heavy metals. Higher valence manganese oxide pair H + And the affinity of the multivalent cations is much greater than the affinity for the alkali metal ions. Thus, the charge characteristics and colloidal stability of the microbial-induced biomanganese oxide are determined by H + Concentration of ions and multivalent metal ions. The manganese oxide has certain oxidation and adsorption capacity to trivalent arsenic, arsenic (III) is adsorbed on the surface of manganese oxide, the trivalent arsenic on the surface can be oxidized into pentavalent arsenic, and As (V) is subjected to coordination reaction on the surface of the manganese oxide to form As (V) -MnO 2 Bidentate dinuclear bridged complexes. Compared with inorganic manganese oxide, the biological manganese oxide has higher specific surface area and higher adsorption efficiency on arsenic.
Therefore, the invention provides a method for restoring arsenic-polluted soil by biological manganese oxide, which can convert exchangeable arsenic in the soil into residual arsenic under the action of bacterial strain metabolites, thereby reducing the mobility and bioavailability of the arsenic in the soil and effectively fixing the arsenic in the soil.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments, and that the above-described embodiments and descriptions are only preferred embodiments of the present invention, and are not intended to limit the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (3)
1. A method for in-situ remediation of arsenic in soil by bacteria is characterized by comprising the following steps: the method comprises the following steps:
s11: adding bacterial liquid capable of inducing to produce biological manganese oxide material into arsenic-polluted soil, and sequentially adding a carbon source and a divalent manganese component;
s12: regulating the water content of the soil to 20-40%, stirring for 10-30min by a stirrer, and then culturing for 2-3 weeks at room temperature to enable the biological manganese oxide material to have physical-chemical reaction with trivalent arsenic or pentavalent arsenic in the soil so as to finish the repair of the arsenic in the soil;
the preparation method of the bacterial liquid capable of inducing the production of the biological manganese oxide material comprises the following specific steps: inoculating the strain into 17-25g/L nutrient broth culture medium for enrichment culture;
the conditions of enrichment culture are as follows: aerobic enrichment culture is carried out for 3-5 days at 15-30 ℃ under horizontal oscillation of 100-180 rpm; in the enrichment culture process, the transfer quantity of the strain is 2-10% according to the volume ratio;
the step S11 specifically includes: adding bacterial liquid which is obtained by enrichment culture and can induce to produce biological manganese oxide material into 50g arsenic-polluted soil, sequentially adding 16mg of peptone, 4mg of yeast extract powder, 2mg of monopotassium phosphate, 4mg of magnesium sulfate, 4mg of sodium nitrate, 2mg of calcium chloride, 2mg of ammonium chloride, 20mg of ferric ammonium citrate, 4mg of manganese sulfate and 2mg of ammonium carbonate; the adding amount of the bacterial liquid is 0.8-1.5mL per gram of soil according to the liquid-solid ratio;
the strain adopts a strain for converting divalent manganese into tetravalent manganese oxide, the strain is specifically Acinetobacter bieleisii WHW-2, and the separation and purification process of the strain is specifically as follows:
domestication: adding a water sample with mixed microbiota into a nutrient broth culture medium, culturing for 72 hours, transferring bacterial liquid in the nutrient broth culture medium into a PYCM culture medium, and continuously domesticating; the composition of the PYCM medium was: 0.8g/L peptone, 0.2g/L yeast extract, 0.1g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L sodium nitrate, 0.1g/L calcium chloride, 0.1g/L ammonium chloride, 1g/L ferric ammonium citrate, 0.2g/L manganese sulfate and 0.1g/L ammonium carbonate;
step-by-step dilution: the bacterial liquid in PYCM is absorbed according to 10 -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 ，10 -6 ，10 -7 ，10 -8 Is diluted in a gradient;
coating: pouring the flat plate to solidify, pouring the bacterial liquid onto the flat plate, and rapidly coating the surface of the flat plate back and forth by using a coater to uniformly distribute the bacterial liquid and grow uniform single bacterial colonies;
plate scribing: preparing a PYCM solid plate, picking a single colony by an inoculating loop, streaking the single colony onto the solid plate, repeating the streaking for a plurality of times to obtain a pure colony, and identifying and naming the acinetobacter bauhini WHW-2 by a pedigree.
2. The method for in situ remediation of arsenic in soil by bacteria of claim 1 wherein: the water content of the soil is regulated to 25%, and the soil is stirred by a stirrer for 25min and then is cultured for 3 weeks at room temperature.
3. Use of the method for in situ remediation of arsenic in soil by bacteria according to any one of claims 1 to 2 in environmental remediation engineering.
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| CN105331551A (en) * | 2014-08-08 | 2016-02-17 | 华中农业大学 | Manganese-oxidized pseudomonas T34, method for preparing biogenic manganese oxide and application of pseudomonas or biogenic manganese oxide in degrading ciprofloxacin |
| CN109277404A (en) * | 2018-11-28 | 2019-01-29 | 青岛理工大学 | A kind of method of bacterium in-situ immobilization As polluted soil |
| CN113020249A (en) * | 2021-03-17 | 2021-06-25 | 中国科学院新疆生态与地理研究所 | Method for repairing soil arsenic pollution in arid region by using SAP (super absorbent polymer) reinforced manganese oxidizing bacteria |
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| CN101429486A (en) * | 2008-11-27 | 2009-05-13 | 华中农业大学 | Brood cell bacillus MK3-1 for purification of heavy metal pollution and uses thereof |
| CN105331551A (en) * | 2014-08-08 | 2016-02-17 | 华中农业大学 | Manganese-oxidized pseudomonas T34, method for preparing biogenic manganese oxide and application of pseudomonas or biogenic manganese oxide in degrading ciprofloxacin |
| CN109277404A (en) * | 2018-11-28 | 2019-01-29 | 青岛理工大学 | A kind of method of bacterium in-situ immobilization As polluted soil |
| CN113020249A (en) * | 2021-03-17 | 2021-06-25 | 中国科学院新疆生态与地理研究所 | Method for repairing soil arsenic pollution in arid region by using SAP (super absorbent polymer) reinforced manganese oxidizing bacteria |
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