CN104630097B - A kind of acidophilus sulfate reduction bacteria strain and its application - Google Patents
A kind of acidophilus sulfate reduction bacteria strain and its application Download PDFInfo
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- CN104630097B CN104630097B CN201410810933.7A CN201410810933A CN104630097B CN 104630097 B CN104630097 B CN 104630097B CN 201410810933 A CN201410810933 A CN 201410810933A CN 104630097 B CN104630097 B CN 104630097B
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Classifications
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Abstract
A kind of method the invention discloses acidophilus sulfate reduction bacteria strain FKB and its for being acidified control.The acidophilus sulfate reduction bacteria strain FKB, spore bacterium Desulfospor nosinus sp. are bent for desulfurization, China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) is preserved on November 27th, 2014, deposit number is：CGMCC No.10072.Liquid microbial inoculum will be made after the fermented culture of the bacterial strain, it is used after simply being improved to soil lime, chicken manure, phosphate fertilizer etc. with plant stability technical tie-up, can acidification control effectively be carried out to Mining Wasteland such as refuse dump, Tailings Dam soil, achieve the purpose that contain acidification, zoology green-recovery, mine reclamation.
Description
Technical field
Screening and application field the invention belongs to microorganism are specifically related to a kind of acidophilus sulfate reduction bacteria strain,
And its method for being acidified control.
Background technology
The soil acidification problem that Non Ferrous Mineral Industry discards ground is all widely present in worldwide.China is as tradition
Mining powers, possess more than 100,000 seat mines at present, continual mining activity produces the huge Mining Wasteland of area, and
Wherein most of Mining Wastelands are not dealt carefully with, and serious soil acidification problem causes entire ecological environment huge
Big destruction, not even a blade of grass grows for acidized area, and the discharge of toxic heavy metal sour water is serious.The main reason of these Mining Wastelands acidification
It is：Most of non-ferrous metal ore bearing strates have various types of metal sulfides (predominantly FeS2), and these metal sulfides
With recovery activity be exposed in air, will a large amount of sulfuric acid be generated by the dioxygen oxidation in air immediately.Ore deposit
The soil acidification that industry discards ground can bring a series of social and environmental problems, and the heavy metallic poison including severe, pollution are neighbouring cultivated
Field soil and neighbouring water body are so as to cause people's health serious threat etc..Therefore, Mining Wasteland acidification problem is
The important problem of entire mining industry urgent need to resolve.
For at present, the acidification control technology of Mining Wasteland mainly includes Physical-separation Technology, chemical neutralization technology, plants
Object stabilization technique and microbiological treatment technology etc..Traditional acidification control technology such as Physical-separation Technology, chemical neutralization technology
The progress of acidization can only be slowed down in limited range, can not achieve the effect that effect a permanent cure, and cost is very high, expended huge
Greatly；Meanwhile efficiently using for these Mining Wasteland land resources is often limited after administering, waste valuable soil
Resource.Plant stability technology is current using wider acidification control technology, it forms in table soil and consume by quickly establishing vegetation
Oxygen layer reduces oxygen and is spread to deeper soil, hinders the oxidation of metal sulfide, so as to achieve the purpose that control acidification.But
Since the soil of Abandoned Land of Mine often lacks the various nutrients needed for plant growth, and most of plant all cannot be
It is grown under strong acid condition, so this phytoremediation technology will often coordinate other measures such as chemistry to neutralize, add all kinds of nutrition
Element etc. could obtain preferable acidification control effect.
Microbiological treatment technology has at low cost, non-secondary pollution and can be from source for other several technologies
The advantages that reaching control soil acidification on head, iron-reducing bacteria (FRB) and sulfate are used in passing research and application more
Reducing bacteria (SRB) handles acidification problem.FRB and SRB can be given birth to by ferrimanganic reduction, sulfate reduction, methane
Into effect and denitrification consumption alkalescent substance generation highly basic, so as to the acidic materials in further sweetening of the soil, reach
To the effect of control acidification；Also, SRB can also be by sulfate reduction, by SO4 2-Reduction generation H2S, and can by into
Single step reaction generates simple substance S or with other heavy metal ion such as Cu2+Deng with reference to and precipitate, meanwhile, sulfate reduction is anti-
Should during also can H present in consumption system+, control acidification is thus achieved the purpose that from source.
SRB be one kind come in every shape, nutrient type it is various, can oxidizing organic substrates or H in anaerobism or micro- oxygen environment2,
And sulphate reducing or other oxysulfides generate H2The bacterium of S or ancient bacterium.It is widely present in soil, rice soil, animal intestine
In road, hot spring, ocean, acid wastewater in mine, petroleum pipeline and the natural gas well.SRB utilizes SO4 2-As final electron acceptor
Come some simple organic matters of degrading, and the H present in consumption system in metabolic process+, generate the H of high concentration2S and
CO2, so as to control acidification.At present, it has been found that SRB belong to up to more than 60, more than 220 plant, pass through 16S rRNA genes in database
SRB points can be 4 main monoids by the Molecular Evolutionary Analysis of sequence：The mesophilic sulfate reducing bacteria of Gram-negative, leather are blue
Family name's positive production gemma sulfate reducing bacteria, sulphate-reducing thermophilic bacterium and sulphate-reducing thermophilic Gu bacterium.However, in nature
Most SRB are neutrophilic, and optimum pH is 6~8 or so, and in pH<Growth is suppressed when 5, is thus limited
Application of the sulfate reducing bacteria in control is acidified is made.But researcher has found that sulfate-reducing process can also be happened at acidity
In environment, this indicates that the microorganism that can realize sulfate reduction at low ph conditions in the presence of one kind, i.e. acidophilus sulfuric acid
Salt reducing bacteria (aSRB).But all the time, the SRB for being separately cultured pure acidophilus (or acidproof) ends in failure, until
1999, Sen＆Johnson just isolated the gram sun that relatively Desulfotomaculun belongs in three plants of morphological features
Property bacterium.However, up to now, SRB only more than ten strains for the acidophilus (acidproof) delivered understand its work(by genome sequencing
Only 2 plants of energy.
In the patent delivered in the past, most of is that sulfate reducing bacteria is used for heavy metal-containing wastewater treatment field
(CN101628773A, CN101195859A etc.), some accidental patents are using sulfate reducing bacteria for Mining Wasteland acid
Change control to repair, but often there is the Bacterial community that uses is unknown, acid resistance, repairing effect be not or not bacterial strain for these technical solutions
Stablize, rest on laboratory stage mostly, practical extensive a series of problems, such as repairing cannot be adapted to very well.For example, patent
CN101037268A discloses " a kind of method of restoring mine entironment ", and sulfate reducing bacteria is used for the acid of Tailings Dam
Change administer, technical solution be generated in mine development tailing, barren rock, metallurgical slag, beneficiation wastewater, smelting wastewater, acidity
Pit water and Ore stockpile leaching water etc. are focused in Tailings Dam, while add sludge and the organic matter that can be degraded by microorganisms,
An anaerobic environment is artificially built in Tailings Dam, under the action of microorganism and sulfate reducing bacteria, sulphion is generated and makes
The pH value of water rises in Tailings Dam, and sulphion precipitation cures various heavy metal ion to prevent its migration.But this method uses
Sulfate reducing bacteria derive from the Natural Selection of sludge, there are Bacterial community described above is unknown, bacterial strain not acid resistance, repair
Multiple effect instability problem；Patent inventor also only simulates related experiment indoors, is not amplified to practical tailing
It is verified in library, can not ensure the effect in practical application；Meanwhile this method needs accumulation to handle, it is impossible to suitable well
The Tailings Dam in Amenorrhea library, also be easy to cause Tailings Dam structural instability, the risk for easily having mud-rock flow；Finally, the technical side
Case can not also realize zoology green-recovery, land reclamation effect, before follow-up phase can not be consolidated be acidified control obtain achievement.
Invention content
It is an object of the invention to overcome prior art cost excessively high, the Bacterial community used is unknown, bacterial strain not acid resistance,
Repairing effect is unstable, rests on laboratory stage mostly, cannot adapt to practical extensive reparation very well, it is impossible to be controlled from source
The shortcomings of mine soil acidification processed, provide a kind of novel acidophilus sulfate reduction bacteria strain.
In order to solve the above technical problems, the present invention is realized by following technical solution.
The invention discloses a kind of acidophilus sulfate reduction bacteria strain FKB, and spore bacterium is bent for desulfurization
It is general to be preserved in China Committee for Culture Collection of Microorganisms on November 27th, 2014 by Desulfospornosinus sp.
Logical microorganism center (abbreviation CGMCC), deposit number are：CGMCC No.10072.
The invention also discloses a kind of making sides of the liquid microbial inoculum containing the acidophilus sulfate reduction bacteria strain FKB
Method includes the following steps：
1) FKB strains are inoculated intoEnriched mediumIn cultivated, nitrogen, which is blown, to be positioned over 30 DEG C of incubators after 20min and stands
Culture becomes prepared Chinese ink color to culture solution from clarifying；
2) cultured bacterium solution is seeded to by 5% inoculum concentration in 10L seed fermentation tanks, leads to nitrogen 20min, 30 DEG C of cultures
Become prepared Chinese ink color to culture solution from clarifying；
3) zymotic fluid in seed fermentation tank is seeded to by 15% inoculum concentration in fermentation tank, leads to nitrogen 30min, fermentation
Condition is：, ferment and become prepared Chinese ink color to culture solution；
4) liquid bacterial agent after fermentation, is made in zymotic fluid pack, ensures that bagging process does not introduce air.
Further, the enrichment culture based formulas in the step 1) is：(NH4)2SO40.45g, KCl0.05g,
MgSO4·7H2O 0.5g, KH2PO40.05g, Ca (NO3)2·4H2O 0.014g, yeast extract 0.2g, glycerine 0.92g steam
Distilled water 1000mL, with 1M H2SO4PH to 3.5 is adjusted, 121 DEG C of high pressure sterilization 20min are added after cooling by filtration sterilization
FeSO4·7H2O 0.5g, ascorbic acid 0.02g, mercaptoethanol 1g.
Further, the culture medium prescription (w/v) in the step 2) is：Cane molasses 5%, yeast leachate 2%, Portugal
Grape sugar 0.5%, (NH4)2SO44.5%, MgSO4·7H2O 5%, KCl 0.5%, KH2PO40.5%, Ca (NO3)2·4H2O
0.14%, FeSO4·7H2O 5%, ascorbic acid 0.5%, pH 5.2.
The production method of liquid microbial inoculum according to claim 2, which is characterized in that ferment described in the step 3)
Condition is：It 30 DEG C, does not stir, pH 5.2, tank presses 0.05~0.07Mpa；Fermentation tank culture based formulas (w/v) is：Cane molasses
10%, yeast leachate 2%, (NH4)2SO44.5%, MgSO4·7H2O 5%, KCl 0.5%, KH2PO40.5%, Ca
(NO3)2·4H2O 0.14%, FeSO4·7H2O 6%, ascorbic acid 0.5%, pH 5.2.
Present disclosure further includes the acidophilus sulfate reduction bacteria strain FKB, the application in soil acidification is controlled.
Further, the application is：Liquid microbial inoculum is made in the acidophilus sulfate reduction bacteria strain FKB, right
Soil lime, chicken manure, phosphate fertilizer etc. use after simply being improved with plant stability technical tie-up.Its specific steps are：
1) bar ditch are excavated in soil surface；
2) lime, chicken manure, phosphate fertilizer being added in order to improve soil matrix, the phosphate fertilizer additive amount is 30g/m2,
It is artificial uniformly to sow the depth for retaining bar ditch 20cm or so to surface, both sides earthen backfill, it is kept for 1 month；
3) it is added in bar ditch according to the FKB microbial inoculum dosages of 15kg per acre, is slightly compacted with both sides earthen backfill, is acted on 14 days；
4) in addition plant species is sowed in bar ditch gap using fish-scale pit plum blossom point arrangement kind plant hole, planting plants nutrient bag seedling
Son and soil seed bank, soil seed pool, cladding thickness 4cm or so straw, guarantee have good ventilation environment；
5) it fosters：It need to be fostered altogether during plantation three times, first time main contents include checking survival rate, ridging, and carry out
After-culture；Second of content includes loosening the soil, expands cave plus lime, applies NPK composite fertilizers, finds dead strain after-culture immediately；Third time is fostered,
Main contents include adding lime, loosen the soil, expand cave, ridging, desinsection, and often spread manuer in holes top dressing NPK composite fertilizer 100g, and cooperation loosens the soil, earths up,
In fertilizer shallow embedding soil, if not raining for a long time, suitably water.
Further, in the step 2), lime used is white lime；Chicken manure used is band husk chicken manure, uses advance
Row sterilization；Phosphate fertilizer used is calcium superphosphate.
Acidophilus sulfate reducing bacteria strain FKB, the deposit number CGMCC No.10072 of the present invention, by all mouth Pb-Zn deposits tails
It is isolated and purified in ore deposit bottom storehouse mud sample product, show that it belongs under Firmicutes by the comparison of 16S rRNA gene orders
Desulfosporosinus belong to.By using conventional Gram's staining, spore staining and scanning electron microscopic observation, as a result table
Bright FKB colony edges are irregular, rod-shaped, belong to sporiferous Gram-negative bacteria, and size is 3.0~6.5 × 0.4~0.7 μm.
Subsequent bio-chemical characteristics detection shows that the physio-biochemical characteristics of FKB are as follows：Growth temperature range is 10~40 DEG C, most suitable
Suitable growth temperature is 30~35 DEG C；The pH of growth ranging from 3.6~6.0, the pH of optimum growth is 5.2；The NaCl of growth
Concentration range is 0~1.8% (w/v), and the NaCl concentration of optimum growth is 0.6%.FKB can utilize sulfate, sulphite,
As unique sulphur source and electron acceptor reduction reaction or disproportionated reaction generation H occur for thiosulfate and elemental sulfur2S is simultaneously generated
Energy supplies thalli growth.As (V) can also be reduced to As (III) by FKB by the use of arsenate as electron acceptor, and As (III) energy
Cell is discharged under specific transporters effect, so as to play the role of reducing cellular toxicity.Nitrate also can conduct
Electron acceptor provides the energy needed for growth for FKB.We are further by genome sequencing technology to the thio of the bacterium
Thank and to the stress response mechanism of environment such as low pH, high heavy metal ion etc. a series of acidifications control relevant critical function into
It has gone and has studied and understand, whereby, we can preferably use it for the acidification control of Mining Wasteland soil.
The acidophilus sulfate reducing bacteria strain FKB, is screened separating obtained by following steps：
After Tailings Dam acquisition bed mud sample, sample collection, enrichment culture is carried out using special enriched medium first,
Culture medium of the culture medium of enrichment culture aSRB with reference to used in Sen and Johnson (1999) culture sulfate reducing bacterias, and according to
Actual conditions do corresponding improvement, and enrichment process is as follows：1g samples to be separated is taken to be placed in the anaerobism bottle of the 150mL to sterilize in advance,
120mL enriched mediums are added in, with high-purity nitrogen stripping 20min to remove the O of culture medium2, by culture after air-blowing
It is placed in 30 DEG C of constant incubators and is protected from light quiescent culture, until culture solution becomes prepared Chinese ink color from clarifying, 5% culture solution is taken to transfer
Into fresh enriched medium, turn training 10 times to remove most heterotrophicy bacteria；
It after the enriched culture of sample, is isolated and purified using the isolation medium of improvement, filters out new acidophilus sulfuric acid
Salt restores bacteria strain, and it is as follows to isolate and purify process：Isolation medium is configured, heat preservation is at 50 DEG C or so after sterilizing, by enrichment culture
Bacterium solution afterwards is diluted to 10-2~10-6The bacteria suspension of concentration takes the bacterium solution of 50 μ L difference dilutions in culture dish, immediately respectively
It pours into isolation medium and gently shakes culture dish, bacterium solution with culture medium is uniformly mixed while hot, is poured into again after agar solidification
Then culture dish is put into the crisper added with anaerobic gas generation bag (Mitsubishi) and carries out anaerobism in 30 DEG C by identical culture medium
It cultivates, about 2 weeks or so, the bacterium colony of appearance black is grown in culture dish, picking single bacterium colony is transferred to anaerobism in fluid nutrient medium and trains
It supports, steps be repeated alternatively until that the colonial morphology grown in culture dish is consistent, you can isolated SRB is identified, is protected
It deposits.
The method for screening and separating, the culture medium prescription used are：
Enriched medium：(NH4)2SO40.45g, KCl 0.05g, MgSO4·7H2O 0.5g, KH2PO40.05g, Ca
(NO3)2·4H2O 0.014g, yeast extract 0.2g, glycerine 0.92g, distilled water 1000mL, with 1M H2SO4Adjust pH to
3.5,121 DEG C of high pressure sterilization 20min, add the FeSO by filtration sterilization after cooling4·7H2O 0.5g, ascorbic acid
0.02g, mercaptoethanol 1g；
Isolation medium：Corresponding drug is weighed by enrichment culture based formulas, is dissolved in 700mL distilled water, with 1M H2SO4It adjusts
Save pH to 3.5；Separately 1g agar is weighed to be dissolved in 300mL distilled water, sterilize 20min respectively at 121 DEG C, is cooled to 50 DEG C or so,
Two parts culture medium is uniformly mixed, adds the FeSO of filtration sterilization4·7H2O 0.5g, ascorbic acid 0.02g, mercaptoethanol
1g。
Compared with prior art, the present invention has the following advantages：
1st, the acidophilus sulfate reducing bacteria strain FKB bacterial strains that the present invention filters out, it belongs to acidophilus sulfate reducing bacteria bacterium
Strain, the aSRBs that the whole world is isolated at present only more than ten strains not only overcome the passing uncertainty using microorganism species, and
And oxyphilous feature also allows the bacterial strain more to can adapt to extreme acid condition, breeding is faster, metabolism is more prosperous, effect is more preferable；Together
When, we have done genome sequencing to FKB, its metabolism and function have been understood in depth, especially in sulfate reduction and anti-pH
The mechanism these two aspects of stress, this can greatly help us preferably to cultivate and controlled using the bacterial strain for being acidified.
2nd, control soil acidification technology of the invention, carries out acidification control using sulfate reducing bacteria, is discarded from mining industry
Ground acidification root inhibits the generation of acidification, while steady with plant after simply being improved to soil lime, chicken manure, phosphate fertilizer etc.
Determine technical tie-up use, avoid the potential threat that after-souring occurs after repair for similar technique, needed for repairing again
A large amount of manpower and materials and the wasting of resources, while can also achieve the purpose that zoology green-recovery, mine reclamation.
3rd, control soil acidification technology of the invention has the characteristics that safety and environmental protection, environmental-friendly, without to original landforms
Large area transformation is carried out, without the soil moved in improve the original for carrying out large area, avoids the destruction fetched earth to periphery mountain forest ecological environment.It is all
Raw material and entire repair process all will not bring secondary pollution to environment.
4th, control soil acidification technology of the invention has the characteristics that of low cost, easy to operate, and in Yongping copper
Ore deposit refuse dump, Ores At Chengmenshan Copper Mine Tailings Dam have carried out large area practical application and have achieved good effect, and acidification phenomenon obtains
Apparent control, vegetation coverage are suitble to promoting the use of for large area more than 90%.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Description of the drawings
Fig. 1 is Gram's staining (1,000 times of optical microphotographs of the acidophilus sulfate reducing bacteria strain FKB bacterial strains of the present invention
Mirror) and scanning electron microscopic observation aspect graph.
The acidophilus sulfate reduction bacteria strain FKB of the present invention is bent spore bacterium Desulfospornosinus sp. for desulfurization,
China Committee for Culture Collection of Microorganisms's common micro-organisms center has been preserved on November 27th, 2014 (referred to as
CGMCC), deposit number is：CGMCC No.10072.
Specific embodiment
Embodiment 1FKB strain isolations are identified and function parsing
1) bacterial screening
From the bed mud sample of all No. 1 Tailings Dam edge collectings of mouth Pb-Zn deposits, collected with sterile 50mL centrifuge tubes, then put
It transports laboratory back in ice bath, is stored in 4 DEG C of refrigerators immediately.
After sample collection, enrichment culture is carried out using special enriched medium first, it is therefore an objective to script be allowed to prevent take up excellent
The sulfate reduction flora of gesture status is able to amount reproduction, and removes most heterotrophicy bacteria.The culture of enrichment culture aSRB
Culture medium of the base with reference to used in Sen and Johnson (1999) culture sulfate reducing bacterias, and done according to actual conditions and accordingly changed
It is good.The ingredient of enriched medium is：(NH4)2SO40.45g；KCl0.05g；MgSO4·7H2O 0.5g；KH2PO40.05g；Ca
(NO3)2·4H2O 0.014g；Yeast extract 0.2g；Glycerine 0.92g, distilled water 1000mL, with 1M H2SO4Adjust pH to
3.5,121 DEG C of high pressure sterilization 20min, add the FeSO by filtration sterilization after cooling4·7H2O 0.5g, ascorbic acid
0.02g, mercaptoethanol 1g.Enrichment process is as follows：1g samples to be separated is taken to be placed in the anaerobism bottle of the 150mL to sterilize in advance, are added
Enter 120mL enriched mediums, with high-purity nitrogen stripping 20min to remove the O of culture medium2.Culture is put after air-blowing
Quiescent culture is protected from light in 30 DEG C of constant incubators, until culture solution becomes prepared Chinese ink color from clarifying, shows sulfate reducing bacteria
Amount reproduction.5% culture solution is taken to be forwarded in fresh enriched medium, it is 10 times thin to remove most heterotrophism to turn training
Bacterium.
It after the enriched culture of sample, is isolated and purified using the isolation medium of improvement, filters out new acidophilus sulfuric acid
Salt restores bacteria strain.The formula of isolation medium is as follows：Corresponding drug is weighed by enrichment culture based formulas, is dissolved in 700mL distillations
In water, with 1M H2SO4Adjust pH to 3.5；Separately 1g agar is weighed to be dissolved in 300mL distilled water, sterilize 20min respectively at 121 DEG C,
50 DEG C or so are cooled to, two parts culture medium is uniformly mixed, adds the FeSO of filtration sterilization4·7H2O 0.5g, Vitamin C
Sour 0.02g, mercaptoethanol 1g.It is as follows to isolate and purify process：Isolation medium is configured according to the method described above, heat preservation is 50 after sterilizing
DEG C or so, the bacterium solution after enrichment culture is diluted to the bacteria suspension of 10-2~10-6 concentration, takes 50 μ L difference dilutions respectively
Bacterium solution pours into isolation medium and gently shakes culture dish, be while hot uniformly mixed bacterium solution with culture medium immediately in culture dish,
It pours into identical culture medium again after agar solidification, then culture dish is put into added with the fresh-keeping of anaerobic gas generation bag (Mitsubishi)
In box Anaerobic culturel is carried out in 30 DEG C.About 2 weeks or so, the bacterium colony of appearance black is grown in culture dish, picking single bacterium colony is transferred to
Anaerobic culturel in fluid nutrient medium.It steps be repeated alternatively until that the colonial morphology grown in culture dish is consistent, you can to detaching
To SRB identified, preserved.
2) strain idenfication and function parsing
Molecular biology identification：After isolated FKB, extracting genome DNA is carried out to it, is expanded using 27F/1492R
16s rRNA gene orders are sent to Hua Da gene after pcr product purifications and are sequenced, obtain its sequence information.Utilize NCBI
The 16S rRNA sequence informations of measured FKB bacterial strains and GenBank database sequences are carried out tetraploid rice, knot by BLAST
Fruit show FKB belong to Clostridiales mesh, Peptococcaceae sections, Desulfosporosinus belong to it is (micro- in the category
Biology all has the function of sulfate reduction).
Morphological features are identified and physio-biochemical characteristics：The form of thalline is using conventional Gram's staining, gemma dye
Color and scanning electron microscopic observation.As a result it is：FKB colony edges are irregular, rod-shaped, belong to sporiferous Gram-negative bacteria, size
It is 3.0~6.5 × 0.4~0.7 μm.After testing, the physio-biochemical characteristics of FKB are as follows：Growth temperature range is 10~40 DEG C, most
Suitable growth temperature is 30~35 DEG C；The pH of growth ranging from 3.6~6.0, the pH of optimum growth is 5.2；Growth
NaCl concentration ranging from 0~1.8% (w/v), the NaCl concentration of optimum growth is 0.6%.FKB can utilize sulfate, sulfurous
As unique sulphur source and electron acceptor reduction reaction or disproportionated reaction generation H occur for hydrochlorate, thiosulfate and elemental sulfur2S is simultaneously
It generates energy and supplies thalli growth.As (V) can also be reduced to As (III) by FKB by the use of arsenate as electron acceptor, and As
(III) cell can be discharged under specific transporters effect, so as to play the role of reducing cellular toxicity.Nitrate
It can provide growth required energy for FKB as electron acceptor.
Full-length genome measures：In order to more clearly understand the acidification controlling mechanism of FKB, we have carried out full-length genome to it
Sequencing.The genomic DNA of FKB is extracted first, and length is then broken into as 500 Hes by Covaris sonicators at random
The segment of 800bp repairs through end, adds sequence measuring joints, purifying, the preparation in PCR amplification completion library.The library built utilizes
250 sequenators of IlluminaMiseq PE carry out it two generation high-flux sequences.Sequencing data is spelled by quality control, sequence
It connects, the draft genome for splicing gained is then subjected to predictive genes, and opened what is obtained using BLASTx by Genemark
It puts the functional databases such as reading frame (ORF) and NCBI-nr (Non redundant), KEGG, egg-NOG and compares progress function note
It releases；Meanwhile predicted gene is uploaded into KAAS and carries out functional annotation, and pass through the basic metabolism (generations such as C, N, P, S to each bacterial strain
Thank) and resistance (including heavy metal, pH resistances and oxidative stress etc.) response mechanism build its metabolic pathway.
The Genome Size of FKB is 5.1Mbp, is made of 40 scaffold, and G+C ratios are 41.8%；Its 16s rDNA
Sequence is as shown in sequence table Seq ID No.1.From the point of view of 16S rRNA systematic growths, with cls gene group bacterial strain
Desulfosporosinus acidiphilus SJ4 are more similar, similarity 96%.We detect volume in FKB
Full gene needed for code catalysis sulfate, sulphite and thiosulfuric acid salt metabolism.First, into intracellular SO4 2Through sulphur
Hydrochlorate adenylase (sat, sulfate adenylyltransferase) catalysis forms APS；Then, it is compiled by aprAB genes
Adenosine sulfate reduction enzyme (adenylylsulfate reductase) the catalysis APS generations SO of code3 2-, eventually by dsrAB bases
Because the sulfate reduction enzyme (dissimilatory sulphate reductase) of coding alienation is catalyzed SO3 2-Generate H2S.And
And the phsA genes detected in FKB genomes can encode thiosulphate reduction enzyme (thiosulfate reductase),
The enzyme can be catalyzed thiosulphate reduction generation H2S.On heavy metal resistance, FKB can go reply to coerce by three kinds of modes：When
The ionic pump that ATP enzyme participates in excludes harmful heavy metal ions, second is that by forming complex compound to reduce heavy metal toxicity, third,
The oxidation reaction of heavy metal cation.In addition, being coerced for low pH, genome sequencing result shows that FKB passes through following four
Mode is responded：1st, pass through Na+/H+ATPase、K+/H+ATPase realizes the exchange discharge of proton；2nd, a series of cytoplasm
Buffer molecule absorbs the proton for penetrating into cell；3rd, ornithine, lysine and arginine decarboxylation consumption proton are catalyzed；4th, it degrades
A variety of organic acids remove proton free.It coerces and rings just because of more than sulfate reduction mechanism, heavy metal resistance mechanism and a variety of pH
The presence of mechanism is answered, FKB, which just has, under the conditions of low pH, high concentration heavy metal ion survives and possess sulphate reducing consumption
H+Control the function of acidification.Whereby, we can be preferably by it on the basis of FKB life habits and function has been understood in detail
It is controlled for the acidification of Mining Wasteland soil.
It is prepared by embodiment 2FKB liquid bacterial agents
1) FKB strains are inoculated into enriched medium and cultivated, using 100mL indigo plant cover glass bottles, nitrogen is needed after inoculation
It blows 20min and removes air, be then placed into 30 DEG C of incubator quiescent cultures and become prepared Chinese ink color to culture solution from clarifying；
2) cultured bacterium solution is seeded to by 5% inoculum concentration in 10L seeding tanks, leads to nitrogen 20min, 30 DEG C of cultures to training
Nutrient solution becomes prepared Chinese ink color from clarifying, and the canned liquid measure of seed fermentation is 70%, and culture medium prescription (w/v) is：Cane molasses 5%, ferment
Female leachate 2%, glucose 0.5%, (NH4)2SO44.5%, MgSO4·7H2O 5%, KCl 0.5%, KH2PO40.5%, Ca
(NO3)2·4H2O 0.14%, FeSO4·7H2O5%, ascorbic acid 0.5%, pH 5.2；
3) zymotic fluid in seed fermentation tank is seeded to by 15% inoculum concentration in fermentation tank, leads to nitrogen 30min, fermentation
Condition is：It 30 DEG C, does not stir, pH 5.2, tank presses 0.05~0.07Mpa, ferments and becomes prepared Chinese ink color, fermentation tank culture to culture solution
Based formulas (w/v) is：Cane molasses 10%, yeast leachate 2%, (NH4)2SO44.5%, MgSO4·7H2O 5%, KCl
0.5%, KH2PO40.5%, Ca (NO3)2·4H2O0.14%, FeSO4·7H2O 6%, ascorbic acid 0.5%, pH 5.2；Not
Declaratives are carried out by fermentation general operation；
4) liquid bacterial agent after fermentation, is made in zymotic fluid pack, ensures that bagging process does not introduce air.
Embodiment 3FKB is for refuse dump acidification control
Place is Jiangxi YONGPING COPPER DEPOSIT south refuse dump, and area is 16800 square metres, is divided into top and side slope Liang great areas
Domain, side slope 10000m2, top 6800m2.The region has following Typical Representative meaning：
(1) side slope is steep, drop is big：According to terrain map of survey and drawing, region cope level 273.3m, slope foot elevation 185.5m,
Height difference reaches 87.8m, and grade of side slope is 1:1~1:Between 1.25.Substantially the landform side slope that can represent refuse dump is steep, drop is big
Situation.
(2) it washes away seriously, erratic boulder is in heaps：Due to for many years and washing away, the regional slope and slope foot account for more than 60% gross area
Essentially erratic boulder, no soil, for plant to survive condition very poor (scene has no any plant growth), belong to refuse dump revegetation
Difficulty compares larger region.
(3) side slope soil property situation is poor：From field condition as can be seen that although still some are native near the region halfpace,
But rarely seen plant growth, scene it is visible have acidification reaction just acutely carry out in, generate hot gas outside emit.
After the prepartion of land of test block, analysis is sampled to entire test block soil, depth selection is 0~20cm, altogether
Obtain pedotheque 96.Analysis result shows：The test block belongs to highly acid, and high Acidification potential exists simultaneously a large amount of free
Acid ion soil；And there is also extremely serious copper ion toxic hazard, subregions for test block lighter lead
Toxicity；Soil nutrient is very deficient, much can not meet macronutrient necessary to plant germination growth.Make a concrete analysis of number
According to see the table below：
Note：NAG units are kg H2SO4/ t, heavy metal index unit are ppm, and nutrient index unit is g/kg.
According to more than soil sample investigation result, it is 12.5kg/m to determine chicken manure additive amount2, white lime additive amount is 7kg/
m2, phosphate fertilizer additive amount is 30g/m2；The laboratory experiment that the refuse dump soil consigned by early period is done is screened and is examined on the spot, really
It is as follows to determine planting scheme：Plant Caulis Miscanthis floriduli, masson pine, locust tree nutrient bag seedling；Festuca Arundinacea, Bermuda grass, paspalum notatum, bubble is broadcast live
The seeds such as paulownia, sesbania, giant knotweed, lettuce fiber crops；Separately plus soil seed bank, soil seed pool, soil seed bank, soil seed pool are derived from local discarded farmland.Concrete operations
Step is：
1) bar ditch are excavated in soil surface；
2) lime, chicken manure, phosphate fertilizer being added in order to improve soil matrix, the phosphate fertilizer additive amount is 30g/m2,
It is artificial uniformly to sow the depth for retaining bar ditch 20cm or so to surface, both sides earthen backfill, it is kept for 1 month；
3) it is added in bar ditch according to the FKB microbial inoculum dosages of 15kg per acre, is slightly compacted with both sides earthen backfill, is acted on 14 days；
4) in addition plant species is sowed in bar ditch gap using fish-scale pit plum blossom point arrangement kind plant hole, planting plants nutrient bag seedling
Son and soil seed bank, soil seed pool, cladding thickness 4cm or so straw, guarantee have good ventilation environment；
5) it fosters：It need to be fostered altogether during plantation three times, first time main contents include checking survival rate, ridging, and carry out
After-culture；Second of content includes loosening the soil, expands cave plus lime, applies NPK composite fertilizers, finds dead strain after-culture immediately；Third time is fostered,
Main contents include adding lime, loosen the soil, expand cave, ridging, desinsection, and often spread manuer in holes top dressing NPK composite fertilizer 100g, and cooperation loosens the soil, earths up,
In fertilizer shallow embedding soil, if not raining for a long time, suitably water.
It marches into the arena at the beginning of test block from 11 months 2013 construction, prepartion of land, base material improvement and addition microbial inoculum work was completed before year
Make, weather, which gets warm again after a cold spell, after the beginning of spring completes planting work, on March 20th, 2014 completes this item purpose whole construction working, switchs to
Later stage fosters work.It on June 9th, 2014 repairs situation We conducted test block and investigates and botanizing work.Through on the spot
It investigates and finds, the acidification situation of test block has been greatly improved, and does not see ongoing acidification reaction, soil battalion
Foster condition significantly improves, and a variety of short herbaceous plant occupy superiority, such as paspalum notatum, Chinese silvergrass, Bermuda grass, the water and soil of script
Wastage has obtained effective containment.Botanizing result show test block share 47 kinds of plant variety, cover draft, shrub,
Arbor, whole coverage is up to 90%, and ensemble average height is 50cm, and riotous growth grows fine.The same year September 11 days, Wo Menjin
Gone second of site inspection and botanizing work.The result shows that test block acidification situation has been clearly better, vegetation is averagely covered
It is 96% to spend, and whole height 160cm, plant entirety growing state is good, has occupied the short draft of superiority originally
Replaced by some tall and big draft, shrub or arbors, such as Caulis Miscanthis floriduli, pale persicaria, ramie, sesbania, Formosan paulownia (Cortex seu Radix Paulowniae kawakamii), it is beneath
Soil property has had more apparent variation, has large increase in water-retaining property, fertility, stability etc..Entire test block
Ecological environment be obviously improved.
Embodiment 4FKB is for Tailings Dam acidification control
Place is Area In Chengmenshan Mining, Jiangxi copper mine chicken claw ditch Tailings Dam, and area is 4000 square metres.Chicken claw ditch Tailings Dam 2006
Bottom has been stopped using, and so far by 6~7 years, tailings drying and consolidating in Tailings Dam, surface checking has certain ground
Base bearing capacity, Tailings Dam reservoir area have largely been dried up, and locally understand ponding in library during rainy season.According to reconnaissance trip, experiment ground surface is put down
Smooth, surface is powder white sand shape, and then closely knit adhesion, hardened phenomenon are very serious for lower floor；Entire sample plot range is without any plant
Growth, surface temperature is high, water shortage, and water retention is poor；Soil layer is soft, and people, which tramples, can leave deeper trace, very unfavorable
In the operation of heavy construction instrument.Carrying out sampling survey work early period, depth selection is 0~20cm, and gross sample number is 50,
Testing index includes acidification three bulk of index, heavy metal index and nutrient index.Sample analysis is the result shows that the Tailings Dam
Test block soil acidification is extremely serious, and pH has higher Acidification potential down to 2.56, and NAG reaches 21.5kg H2SO4/
T, copper ion are poisoned seriously, and there is a serious shortage in the supply for nutrient needed for plant growth.Concrete analysis result see the table below：
Note：NAG units are kg H2SO4/ t, heavy metal index unit are ppm, and nutrient index unit is g/kg.
According to more than soil sample investigation result, it is 15kg/m to determine chicken manure additive amount2, white lime additive amount is 10kg/
m2, phosphate fertilizer additive amount is 30g/m2；It is operated according to technical solution described in embodiment 3.The Tailings Dam soil consigned by early period is done
Laboratory experiment screening and examine on the spot, determine that planting scheme is as follows：Plant Panicum repens, ramie, Siberian cocklebur, locust tree nutrient bag
Seedling；The seeds such as Festuca Arundinacea, Bermuda grass, paspalum notatum, sesbania, pale persicaria are broadcast live；Separately plus soil seed bank, soil seed pool, soil seed bank, soil seed pool are derived from
The discarded meadow in locality.
Project was marched into the arena on January 6th, 2014, on 2 5th, 2014 construction workings for being basically completed experimental project, simultaneously
Work is fostered after constructing after turning.On June 28th, 2014, we have carried out test block botanizing and soil sample analysis.
Botanizing has preliminarily formed various plants matching mutually long growth situation, plant the result shows that test block shares 26 kinds of plant
For average plant height up to 50cm, root system depth in more than 10cm, legume is averaged plant height 20cm, root system depth in more than 20cm, beans
Section plant has formd good rhizobium, enhances plant nitrogen fixing capacity.Vegetation cover degree is up to more than 85%, and each kind root system is
It is crisscross, complementary, the root system network of control tailings earth's surface is formed, microorganism and root system of plant are made jointly in plant substrates
With the foundation of vegetation promotes tailings significantly to accelerate into native trend, and realization control is acidified, the target of zoology green-recovery.Soil sample point
Analysis the result shows that, repair about 5 months after, sample plot pH has risen to 7.90, and highly acid has been effectively improved；NAG-pH
It is 3.38, potential production acid is effectively controlled；EC values are down to 0.95ms/cm by 2.34ms/cm, a large amount of existing free in soil
It poisons ion to have greatly reduced, the acidification situation of entire sample plot has been effectively improved.The project has been led at present
It crosses and passes through Jiangxi Copper Co., Ltd.'s examination.
Claims (9)
1. a kind of acidophilus sulfate reduction bacteria strain FKB, it is characterised in that：The bacterial strain belongs to desulfurization bending spore bacterium
(Desulfosporosinus sp.), deposit number is：CGMCC No.10072.
2. a kind of production method of the liquid microbial inoculum containing acidophilus sulfate reduction bacteria strain FKB described in claim 1, including
Following steps：
1) FKB strains are inoculated into enriched medium and cultivated, 30 DEG C of quiescent cultures of anaerobism become to culture solution from clarifying
Prepared Chinese ink color；
2) cultured bacterium solution is seeded to by 5% inoculum concentration in seed fermentation tank, 30 DEG C of quiescent cultures of anaerobism to culture solution by
Clarification becomes prepared Chinese ink color；
3) zymotic fluid in seed fermentation tank is seeded to by 15% inoculum concentration in fermentation tank, 30 DEG C of quiescent cultures of anaerobism to training
Nutrient solution becomes prepared Chinese ink color；
4) liquid microbial inoculum after fermentation, is made in zymotic fluid pack, ensures that bagging process does not introduce air.
3. the production method of liquid microbial inoculum according to claim 2, it is characterised in that：Enrichment training in the step 1)
Foster based formulas is：(NH4)2SO40.45g, KCl 0.05g, MgSO4·7H2O 0.5g, KH2PO40.05g, Ca (NO3)2·
4H2O 0.014g, yeast extract 0.2g, glycerine 0.92g, distilled water 1000mL, with 1M H2SO4Adjust pH to 3.5,121 DEG C
High pressure sterilization 20min adds the FeSO by filtration sterilization after cooling4·7H2O 0.5g, ascorbic acid 0.02g, sulfydryl second
Alcohol 1g.
4. the production method of liquid microbial inoculum according to claim 2, it is characterised in that：Culture medium in the step 2)
It is formulated as w/v：Cane molasses 5%, yeast leachate 2%, glucose 0.5%, (NH4)2SO4 4.5%, MgSO4·7H2O
5%, KCl 0.5%, KH2PO40.5%, Ca (NO3)2·4H2O 0.14%, FeSO4·7H2O 5%, ascorbic acid 0.5%,
pH 5.2。
5. the production method of liquid microbial inoculum according to claim 2, it is characterised in that：It ferments described in the step 3)
Condition is：It 30 DEG C, does not stir, pH 5.2, tank presses 0.05~0.07Mpa；Fermentation tank culture based formulas is w/v：Cane molasses
10%, yeast leachate 2%, (NH4)2SO4 4.5%, MgSO4·7H2O 5%, KCl 0.5%, KH2PO40.5%, Ca
(NO3)2·4H2O 0.14%, FeSO4·7H2O 6%, ascorbic acid 0.5%, pH 5.2.
6. applications of the acidophilus sulfate reduction bacteria strain FKB described in claim 1 in soil acidification is controlled.
7. application according to claim 6, it is characterised in that：By acidophilus sulfate reducing bacteria bacterium described in claim 1
Liquid microbial inoculum is made in strain FKB, is used after being improved to soil lime, chicken manure, phosphate fertilizer with plant stability technical tie-up.
8. application according to claim 7, step are：
1) bar ditch are excavated in soil surface；
2) it adds lime, chicken manure, phosphate fertilizer in order to improve soil matrix, the phosphate fertilizer additive amount is 30g/m2, it is artificial equal
It is even to sow the depth for retaining bar ditch 20cm to surface, both sides earthen backfill, it is kept for 1 month；
3) it is added in bar ditch according to the FKB microbial inoculum dosages of 15kg per acre, is slightly compacted with both sides earthen backfill, is acted on 14 days；
4) bar ditch gap is using fish-scale pit plum blossom point arrangement kind of a plant hole, planting plants nutrient bag seedling, in addition sow vegetable seeds and
Soil seed bank, soil seed pool, cladding thickness 4cm straw, guarantee have good ventilation environment；
5) it fosters：It need to be fostered altogether during plantation three times, first time main contents include checking survival rate, ridging, and carry out after-culture；
Second of content includes loosening the soil, expands cave plus lime, applies NPK composite fertilizers, finds dead strain after-culture immediately；Third time is fostered, main interior
Hold and include adding lime, loosen the soil, expand cave, ridging, desinsection, often spread manuer in holes top dressing NPK composite fertilizer 100g, and cooperation loosens the soil, earths up, fertilizer
It is appropriate to water if not raining for a long time in shallow embedding soil.
9. application according to claim 8, which is characterized in that in the step 2), lime used is white lime；Chicken used
Excrement is band husk chicken manure, is sterilized before use；Phosphate fertilizer used is calcium superphosphate.
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