CN101586092A - Iron-reducing comamonas and application thereof - Google Patents

Iron-reducing comamonas and application thereof Download PDF

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CN101586092A
CN101586092A CNA2009101466934A CN200910146693A CN101586092A CN 101586092 A CN101586092 A CN 101586092A CN A2009101466934 A CNA2009101466934 A CN A2009101466934A CN 200910146693 A CN200910146693 A CN 200910146693A CN 101586092 A CN101586092 A CN 101586092A
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iron
comamonas
reducing
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周顺桂
武春媛
王弋博
李芳柏
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Guangdong Institute of Eco Environment and Soil Sciences
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Guangdong Institute of Eco Environment and Soil Sciences
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Abstract

The invention discloses an iron-reducing comamonas and an application thereof. The inventor screens the iron-reducing comamonas with the humus reduction activity and the iron reduction activity by a great deal of experiments. The bacteria can reduce the ferric iron such as ironic citrate, iron hydroxide, ferrihydrite and goethite; and reduce the humus and the humic model AQDS (anthraquinone-2, 6-disulfonic acid). The iron-reducing comamonas is the facultative anaerobe, has wide electro donor spectrum. The electro donors which can be oxidized by the bacteria in the anaerobic condition are glycerine, glucose and sucrose.

Description

Iron-reduced tuftedmonas and application thereof
The present invention divides an application, and the applying date of its female case is on June 1st, 2007, and application number is 200710028384.8, and denomination of invention is " iron-reduced tuftedmonas and application thereof "
Technical field
The present invention relates to novel bacterial triage techniques field, specifically, relate to a kind of iron-reduced tuftedmonas and application thereof.
Background technology
Fe (III) and soil ulmin are a large amount of two kinds of components that exist in the soil.Fe (III) mainly exists with the insoluble form of iron oxide, and average content is about 5.6% in the earth's crust, is the highest electron acceptor(EA) of natural abundance in the anaerobism soil.Soil ulmin is to be rich in natural organic mixture quinonyl, heterogeneous, is the main body of the soil organism, accounts for 60～80% of total organic carbon, all has vital role at aspects such as soil fertility, agricultural sustainable development, environment protection.
Soil ulmin reduction is called humus respiration again, is meant that microorganism passes through respiration, with the soil ulmin of transfer transport to the soil, the soil ulmin of oxidation state is reduced into the ortho states soil ulmin, and thalline obtains the process of energy in metabolic process; Studies show that in recent years, soil ulmin can serve as the electron acceptor(EA) of mineralization of organic material in anaerobic environment, quicken the degraded of organic pollutant.Fe (III) reduction claims Fe (III) to breathe again, be meant microorganism by respiration with transfer transport to extracellular ferriferous oxide surface, Fe (III) is reduced into Fe (II), and from this process, obtains the process of energy.The direct result that soil ulmin and Fe (III) breathe is the oxidation state soil ulmin to be converted into go back ortho states, and Fe (III) is converted into Fe (II), for mineralization of organic material and pollutent reduction reparation provide basic driver power.According to estimates, in some flooded soils and freshwater sediment, Fe (III)/humus respiration has directly caused the organic carbon mineralization more than 80%, and its contribution surpasses the summation of other anaerobic metabolism modes such as nitrate respiration, vitriol breathing, methanogenesis.Pollutent biology in situ recovery technique under soil ulmin and Fe (III) reductive condition has become international hot fields.
Soil ulmin and Fe (III) reduction is the enzymatic reaction by the specified microorganisms mediation, and the bacterium with soil ulmin and Fe (III) reducing activity is the key that reaction is carried out.At present, have been found that the strain more than 100 of soil ulmin and Fe (III) reducing bacteria, mainly be distributed in ground Bacillaceae (Geobactersp.), Shiva Bordetella (Shewanella sp.), Desulfobacter (Desulfitobacteriumsp.), Desulfovibrio several genus such as (Desulfomicrobium sp.).These bacterial strains remain in following defective: 1) most of bacterial strains can only be survived in the strictly anaerobic environment, are unfavorable for scale operation and practical application; 2) the electron donor utilization of bacterial strain spectrum is narrower, can only utilize small molecular weight organism such as acetate, lactic acid as electron donor, can not utilize glucose, organism that the sucrose equimolecular quantity is bigger, and is higher relatively to the environmental requirement of practical application.So far do not find to have the comamonas of soil ulmin reduction or Fe (III) reducing activity as yet.
Summary of the invention
The object of the present invention is to provide iron-reduced tuftedmonas with soil ulmin reduction and iron reducing activity.
Another object of the present invention is to provide the application of above-mentioned iron-reduced tuftedmonas in soil ulmin reduction or ferric iron reduction.
The iron-reduced tuftedmonas of separation screening gained of the present invention (Comamonasferrireducens) CY01 is a Gram-negative bacteria, elongated rod shape, in on March 12nd, 2007 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered: CGMCC 1968.
One, the separation of bacterial classification, purifying
Get the ancient forest soil sample of 5g in the 100mL liquid nutrient medium, substratum consists of (g/L): AQDS (anthraquinone-2,6-disulfonic acid) 0.0412g, sodium acetate 1.36g, NaHCO 32.5g, NH 4Cl 0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, (every liter of vitamin solution contains vitamin solution 10.0mL: vitamin H 2.0mg, folic acid 2.0mg, vitamins B 610.0mg, riboflavin 5.0mg, vitamins B 150mg, nicotinic acid 5.0mg, pantothenic acid 5.0mg, vitamins B 120.1mg, Thioctic Acid 5.0mg), trace element solution 10.0mL (contains in every liter of trace element solution: nitrilotriacetic acid(NTA) 1.5g, MgSO 47H 2O 3.0g, MnSO 4H 2O 0.5g, NaCl 1.0g, FeSO 47H 2O 0.1g, CoCl 26H 2O 0.1g, CaCl 20.1g, ZnSO 47H 2O 0.1g, CuSO 45H 2O 0.01g, AlKSO 412H 2O 0.01g, H 3BO 30.01g, Na 2M 0O 40.01g, NiCl 26H 2O 0.024g), pH 5.0～5.5.Drum fills high-purity mixed gas (N in the inoculation forward direction nutrient solution 2/ CO 2=80/20) 〉=1 hour, the aluminium lid sealing, nutrient solution places anaerobism workstation (N 2/ CO 2=80/20), 30 ℃ leave standstill cultivation, detect the colour-change situation of observing nutrient solution when going back ortho states soil ulmin model thing AQDS (anthraquinone-2,6-disulfonic acid) productive rate.When the output of going back ortho states AQDS constantly increased, nutrient solution was from the colourless yellow that becomes, and color is deepened gradually.When the nutrient solution colour stable no longer changes, the inoculum size enrichment culture with 10% three times, dilution is then evenly coated (every liter of nutrient solution adds 16 gram agar, and other composition is with above-mentioned liquid nutrient medium) on the solid medium, places anaerobism workstation (N 2/ CO 2=80/20) cultivated 48 hours for 30 ℃, picking list bacterium colony separates, purifying.
Two, bacterial strain of the present invention has following form and physiology, biochemical characteristic:
(1) thalli morphology characteristic
Adopt conventional bacterium Physiology and biochemistry authentication method and electron microscope observation, this bacterial strain is a Gram-negative bacteria, and elongated rod shape, size are 1.2～1.5 * 0.3～0.4 μ m, have mobility.
(2) colonial morphology characteristic
Aerobic cultivation is after 24 hours on beef extract-peptone agar solid medium flat board, and colonial morphology is circular, and smooth surface is flat, neat in edge, and pale pink, bacterium colony size (d) is 1～2mm; After 48 hours, colonial morphology is circular in aerobic cultivation on the above-mentioned substratum, and smooth surface is flat, neat in edge, and yellow, bacterium colony size (d) is 3～4mm.
(3) main physiology, biochemical characteristic
Oxydase reaction is positive under aerobic condition, aerogenesis not, and Citrate trianion utilization test is positive.Ability with amphimicrobian growth; Under aerobic condition, identify the back discovery with BIOLOG, in 96 kinds of different carbon sources, this bacterium can utilize wherein 38 kinds.Electron donor that under anaerobic can oxidation has: glycerol, glucose, sucrose; Utilize above-mentioned electron donor, this bacterium can reduce ferric irons such as ironic citrate, ironic hydroxide, ferrihydrite, pyrrhosiderite, also can go back prohumic substances and soil ulmin model thing AQDS (anthraquinone-2,6-disulfonic acid).
(4) molecular biological characteristic
Adopt the SDS-Proteinase K, chloroform-primary isoamyl alcohol (volume ratio 24: 1) extracting, the method for 0.6 volume isopropanol precipitating is extracted bacteria total DNA.Adopt the 16S rDNA of bacterial 16 S rDNA universal primer F8 and R1542 amplification bacterium, tangible band appears near 1500bp, to carry out sequencing after the pcr amplification product recovery, dna sequence dna input GenBank with obtaining compares to all sequences in the database with the Blastn program.In conjunction with the result of above-mentioned physio-biochemical characteristics, 16S rDNA sequence, this bacterial strain should belong to Comamonas, called after iron-reduced tuftedmonas (Comamonas ferrireducens) CY01.
Compared with prior art, the present invention has following beneficial effect:
The contriver has filtered out iron-reduced tuftedmonas through a large amount of experimental studies, and it has soil ulmin reduction and iron reducing activity.This bacterium can reduce ferric irons such as ironic citrate, ironic hydroxide, ferrihydrite, pyrrhosiderite; Also can go back prohumic substances and soil ulmin model thing AQDS (anthraquinone-2,6-disulfonic acid).The iron-reduced tuftedmonas amphimicrobian, electron donor utilization spectrum is wider, this bacterium can be under anaerobic can oxidation electron donor have: glycerol, glucose, sucrose.
Description of drawings
Fig. 1 is the scanning electron microscope picture of Comamonas ferrireducens CY01.
Embodiment
Embodiment 1: the separation of bacterial classification, purifying
Get the ancient forest soil sample of 5g in the 100mL liquid nutrient medium, substratum consists of (g/L): AQDS (anthraquinone-2,6-disulfonic acid) 0.0412g, sodium acetate 1.36g, NaHCO 32.5g, NH 4Cl 0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, (every liter of vitamin solution contains vitamin solution 10.0mL: vitamin H 2.0mg, folic acid 2.0mg, vitamins B 610.0mg, riboflavin 5.0mg, vitamins B 150mg, nicotinic acid 5.0mg, pantothenic acid 5.0mg, vitamins B 120.1mg, Thioctic Acid 5.0mg), trace element solution 10.0mL (contains in every liter of trace element solution: nitrilotriacetic acid(NTA) 1.5g, MgSO 47H 2O 3.0g, MnSO 4H 2O 0.5g, NaCl 1.0g, FeSO 47H 2O 0.1g, CoCl 26H 2O 0.1g, CaCl 20.1g, ZnSO 47H 2O 0.1g, CuSO 45H 2O 0.01g, AlKSO 412H 2O 0.01g, H 3BO 30.01g, Na 2M 0O 40.01g, NiCl 26H 2O 0.024g), pH 5.0～5.5.Inoculation is gone to rouse in the nutrient solution and is filled high-purity mixed gas (N 2/ CO 2=80/20) 〉=1 hour, the aluminium lid sealing, nutrient solution places anaerobism workstation (N 2/ CO 2=80/20), 30 ℃ leave standstill cultivation, detect the colour-change situation of observing nutrient solution when going back ortho states soil ulmin model thing AQDS (anthraquinone-2,6-disulfonic acid) productive rate.When the output of going back ortho states AQDS constantly increased, nutrient solution was from the colourless yellow that becomes, and color is deepened gradually.When the nutrient solution colour stable no longer changes, the inoculum size enrichment culture with 10% three times, dilution is then evenly coated (every liter of nutrient solution adds 16 gram agar, and other composition is with above-mentioned liquid nutrient medium) on the solid medium, places anaerobism workstation (N 2/ CO 2=80/20) cultivated 48 hours for 30 ℃, picking list bacterium colony separates, purifying.
Embodiment 2: the evaluation of bacterial classification
(1) mensuration of Physiology and biochemistry character:
The physio-biochemical characteristics of table 1.CY01 bacterial strain
Figure A20091014669300081
Annotate :+expression can utilize;-expression can not utilize
(2) molecular biology identification
Adopt the SDS-Proteinase K, chloroform-primary isoamyl alcohol (volume ratio 24: 1) extracting, the method for 0.6 volume isopropanol precipitating is extracted bacteria total DNA.Adopt the 16S rDNA of bacterial 16 S rDNA universal primer F8 and R1542 amplification bacterium, tangible band appears near 1500bp, to carry out sequencing after the pcr amplification product recovery, dna sequence dna input GenBank with obtaining compares analysis with the Blastn program to all sequences in the database.In conjunction with the result of above-mentioned physio-biochemical characteristics, 16S rDNA sequence, this bacterial strain should belong to Comamonas, called after iron-reduced tuftedmonas (Comamonas ferrireducens) CY01.
Embodiment 3: the evaluation of reducing activity
Electron donor is a glucose, and electron acceptor(EA) is a ferrihydrite
Culture medium prescription: contain ferrihydrite 10g in every liter of deionized water, NaHCO 32.5g, NH 4Cl0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, glucose 0.9008g, each 10.0mL of vitamin solution and trace element solution (vitamin solution and trace element solution prescription are with embodiment 1), pH 5.0～5.5; In 121 ℃ of sterilizations 20 minutes, glucose and other composition separately after the sterilization mixed glucose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5 days.Adjacent phenanthroline method is measured the content of Fe (II), calculates Fe (III) reduction ratio.Fe (III) reduction ratio=Fe (II) concentration/Fe (III) initial concentration * 100%.
Table 2 is electron donor with glucose, and ferrihydrite is an electron acceptor(EA), has verified the iron reducing power of C.ferrireducens CY01 bacterial strain.
Fe (III) reduction ratio (%) under the different incubation times of table 2.
0 day 5 days 10 days 15 days 20 days
Contrast 0.04 0.54 0.87 1.52 2.03
The CY0 bacterial strain 0.04 0.89 2.23 3.42 4.74
Embodiment 4: the evaluation of reducing activity
Electron donor is a glucose, and electron acceptor(EA) is a pyrrhosiderite
Culture medium prescription: contain pyrrhosiderite 9g in every liter of deionized water, NaHCO 32.5g, NH 4Cl0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, glucose 0.9008g, each 10.0mL of vitamin solution and trace element solution (vitamin solution and trace element solution prescription are with embodiment 1), pH 5.0～5.5; In 121 ℃ of sterilizations 20 minutes, glucose and other composition separately after the sterilization mixed glucose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 10 days.Adjacent phenanthroline method is measured the content of Fe (II), calculates Fe (III) reduction ratio.Fe (III) reduction ratio=Fe (II) concentration/Fe (III) initial concentration * 100%.
Table 3 is electron donor with glucose, and pyrrhosiderite is an electron acceptor(EA), has verified the iron reducing power of C.ferrireducens CY01 bacterial strain.
Fe (III) reduction ratio (%) under the different incubation times of table 3.
0 day 10 days 20 days 30 days 40 days
Contrast 0.08 1.14 2.16 2.54 2.84
The CY0 bacterial strain 0.09 1.74 3.36 5.42 6.74
Embodiment 5: the evaluation of reducing activity
Electron donor is a sucrose, and electron acceptor(EA) is a ferrihydrite
Culture medium prescription: contain ferrihydrite 10g in every liter of deionized water, NaHCO 32.5g, NH 4Cl0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, sucrose 3.423g, each 10.0mL of vitamin solution and trace element solution (prescription is with embodiment 1), pH 5.0～5.5; In 121 ℃ of sterilizations 20 minutes, sucrose and other composition separately after the sterilization mixed sucrose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL 0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5 days.Adjacent phenanthroline method is measured the content of Fe (II), calculates Fe (III) reduction ratio.Fe (III) reduction ratio=Fe (II) concentration/Fe (III) initial concentration * 100%.
Table 4 is electron donor with sucrose, and ferrihydrite is an electron acceptor(EA), has verified the iron reducing power of C.ferrireducens CY01 bacterial strain.
Fe (III) reduction ratio (%) under the different incubation times of table 4.
0 day 5 days 10 days 15 days 20 days
Contrast 0.03 1.17 1.22 2.16 3.24
The CY0 bacterial strain 0.04 1.47 1.61 13.42 16.74
Embodiment 6: the evaluation of reducing activity
Electron donor is a sucrose, and electron acceptor(EA) is a pyrrhosiderite
Culture medium prescription: contain pyrrhosiderite 9g in every liter of deionized water, NaHCO 32.5g, NH 4Cl0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, sucrose 3.423g, each 10.0mL of vitamin solution and trace element solution (vitamin solution and trace element solution prescription are with embodiment 1), pH 5.0～5.5; In 121 ℃ of sterilizations 20 minutes, sucrose and other composition separately after the sterilization mixed sucrose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5 days.Adjacent phenanthroline method is measured the content of Fe (II), calculates Fe (III) reduction ratio.Fe (III) reduction ratio=Fe (II) concentration/Fe (III) initial concentration * 100%.
Table 5 is electron donor with glucose, and ferrihydrite is an electron acceptor(EA), has verified the iron reducing power of C.ferrireducens CY01 bacterial strain.
Fe (III) reduction ratio (%) under the different incubation times of table 5.
0 day 5 days 10 days 15 days 20 days
Contrast 0.05 0.99 1.22 1.85 3.25
The CY0 bacterial strain 0.06 1.18 1.57 7.82 13.3
The evaluation of embodiment 7 reducing activities
Electron donor is a glucose, and electron acceptor(EA) is soil ulmin model thing AQDS
Culture medium prescription: contain AQDS 0.0412g, NaHCO in every liter of deionized water 32.5g, NH 4Cl 0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, sucrose 3.423g, each 10.0mL of vitamin solution and trace element solution (vitamin solution and trace element solution prescription are with embodiment 1), pH 5.0～5.5; Sterilization is 20 minutes under 121 ℃ of conditions, and glucose and other composition separately after the sterilization mix glucose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got nutrient solution every 1 day and precipitations such as thalline are filtered,, calculate the reduction ratio of AQDS with the content of ultraviolet spectrophotometer mensuration reduced form AQDS with 0.22 μ m bacterial filter.AQDS reduction ratio=go back ortho states AQDS concentration/AQDS initial concentration * 100%.
Table 6 is electron donor with glucose, and AQDS is an electron acceptor(EA), has verified the soil ulmin reducing power of C.ferrireducens CY01 bacterial strain.
AQDS reduction ratio (%) under the different incubation times of table 6.
0 day 1 day 2 days 3 days 4 days
Contrast 0.10 0.10 0.24 0.21 0.31
The CY0 bacterial strain 0.18 4.3 17.6 66.3 90.1
Embodiment 8: the evaluation of reducing activity
Electron donor is a sucrose, and electron acceptor(EA) is soil ulmin (available from a U.S. Aldrich company)
Culture medium prescription: contain soil ulmin 0.5g in every liter of deionized water, NaHCO 32.5g, NH 4Cl 0.25g, NaH 2PO 42H 2O 0.678g, KCl 0.1g, sucrose 3.423g, each 10.0mL of vitamin solution and trace element solution (vitamin solution and trace element solution prescription are with embodiment 1), pH 5.0～5.5; In 121 ℃ of sterilizations 20 minutes, sucrose and other composition separately after the sterilization mixed sucrose and other composition during sterilization, and drum fills high-purity mixed gas (N then 2/ CO 2=80/20) 〉=1 hour, inoculation C.ferrireducens CY01 bacterial suspension makes its bacterium number reach 10 8/ mL leaves standstill anaerobism in 30 ℃ and cultivates, and the contrast that does not add bacterium is set simultaneously.Got nutrient solution every 1 day and precipitations such as thalline are filtered, measure and go back the ortho states humus content, calculate the reduction ratio of soil ulmin with 0.22 μ m bacterial filter.Soil ulmin reduction ratio=go back ortho states soil ulmin concentration/soil ulmin initial concentration * 100%.
Table 7 is electron donor with sucrose, and soil ulmin is an electron acceptor(EA), has verified the soil ulmin reducing power of C.ferrireducens CY01 bacterial strain.
Soil ulmin reduction ratio (%) under the different incubation times of table 7.
0 day 1 day 2 days 3 days 4 days
Contrast 0.10 0.10 0.13 0.12 0.14
The CY0 bacterial strain 0.174 0.209 21.7 63.2 86.9

Claims (1)

1. iron-reduced tuftedmonas is characterized in that: it is Gram-negative bacteria, elongated rod shape, on March 12nd, 2007 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered: CGMCC 1968.
CNA2009101466934A 2007-06-01 2007-06-01 Iron-reducing comamonas and application thereof Pending CN101586092A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108456649A (en) * 2018-03-14 2018-08-28 中国石油大学(北京) It restores the proteus of Fe (III) in clay mineral and its inhibits clay swell application
CN109626771A (en) * 2018-12-13 2019-04-16 广东省生态环境技术研究所 A method of promote anaerobically digested sludge heavy metal stable

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108456649A (en) * 2018-03-14 2018-08-28 中国石油大学(北京) It restores the proteus of Fe (III) in clay mineral and its inhibits clay swell application
CN108456649B (en) * 2018-03-14 2021-10-29 中国石油大学(北京) Proteobacteria for reducing Fe (III) in clay mineral and application thereof in inhibiting clay swelling
CN109626771A (en) * 2018-12-13 2019-04-16 广东省生态环境技术研究所 A method of promote anaerobically digested sludge heavy metal stable

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