CN104630097A - Acidophilus sulfate reducing bacterium strain and application thereof - Google Patents
Acidophilus sulfate reducing bacterium strain and application thereof Download PDFInfo
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- CN104630097A CN104630097A CN201410810933.7A CN201410810933A CN104630097A CN 104630097 A CN104630097 A CN 104630097A CN 201410810933 A CN201410810933 A CN 201410810933A CN 104630097 A CN104630097 A CN 104630097A
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Abstract
The invention discloses an acidophilus sulfate reducing bacterium strain FKB and a method for acidification control with the strain. The acidophilus sulfate reducing bacterium strain FKB is desulfospornosinus sp, and is preserved at the China General Microbiological Culture Collection Center (CGMCC for short) on November 27, 2014; and the preservation number is CGMCC No.10072. The strain is prepared into a liquid microbial inoculum after fermentation culture; and soil is simply improved with lime, chicken manure, a phosphate fertilizer and the like and then the liquid microbial inoculum can be used together with a plant stabilizing technology to perform the acidification control on soil of mining wastelands such as refuse dumps and tailings ponds; and the targets of acidification restraint, ecological regreening and mine land rehabilitation are reached.
Description
Technical field
The invention belongs to screening and the Application Areas of microorganism, be specifically related to a kind of addicted to sour sulfate reduction bacteria strain, and for method that acidifying controls.
Background technology
The soil acidification problem that Non Ferrous Mineral Industry discards ground all extensively exists in worldwide.China is as traditional mining powers, have more than 100,000 seat mines at present, continual mining activity creates the huge Mining Wasteland of area, and wherein most of Mining Wasteland is not dealt carefully with, serious soil acidification problem causes huge destruction to whole ecotope, not even a blade of grass grows in acidized area, and the discharge of toxic heavy metal sour water is serious.The main reason of these Mining Wasteland acidifyings is: most of non-ferrous metal ore bearing strate has various types of metallic sulfide (to be mainly FeS
2), and these metallic sulfides are exposed in atmosphere along with the carrying out of recovery activity, will be generated a large amount of sulfuric acid immediately by the dioxygen oxidation in air.The soil acidification of Mining Wasteland can bring a series of social and environmental problems, comprises the heavy metallic poison of severe, pollution contiguous cultivated land soil and neighbouring water body thus causes serious threat etc. to the health of people.Therefore, Mining Wasteland acidifying problem is the important problem that whole mining industry needs solution badly.
At present, the acidifying control techniques of Mining Wasteland mainly comprises Physical-separation Technology, chemical neutralization technology, plant stability technology and microbiological treatment technology etc.Traditional acidifying control techniques can only slow down the carrying out of acidization as Physical-separation Technology, chemical neutralization technology in limited scope, can not reach the effect of effecting a permanent cure, and cost is very high, expends huge; Meanwhile, after administering, often limit effective utilization of these Mining Wasteland land resourcess, waste valuable soil resource.Plant stability technology uses wider acidifying control techniques at present, and it is by setting up vegetation fast, forms oxygen consumption layer at table soil, reduces oxygen to more deep soil diffusion, hinder the oxidation of metallic sulfide, thus reach the object controlling acidifying.But the soil due to Abandoned Land of Mine often lacks the various nutritive elements needed for plant-growth, and majority of plant all can not grow under strongly acidic conditions, so this phytoremediation technology often will coordinate other measures as chemical neutralization, add all kinds of nutritive elements etc. and just can obtain good acidifying control effects.
Microbiological treatment technology is for other several technology, have that cost is low, non-secondary pollution and advantages such as controlling soil acidification can be reached from source, in passing investigation and application, adopt iron-reducing bacteria (FRB) and sulphate reducing bacteria (SRB) to process acidifying problem more.FRB and SRB can pass through ferrimanganic reductive action, sulfate reduction, methane generation effect and denitrification and consume weakly alkaline material generation highly basic, thus the acidic substance in further sweetening of the soil, reach the effect controlling acidifying; Further, SRB also by sulfate reduction, by SO
4 2-reduction generates H
2s, and by react further generate simple substance S or with other heavy metal ion as Cu
2+precipitate Deng combination, meanwhile, the H that also can exist in consumption system in sulfate reduction process
+, the object controlling acidifying is so just reached from source.
SRB is that a class comes in every shape, nutrient type is various, can oxidizing organic substrates or H in anaerobism or micro-oxygen environment
2, and sulphate reducing or other oxysulfides generate H
2the bacterium of S or ancient bacterium.It is extensively present in soil, rice soil, animal intestinal, hot spring, ocean, acid wastewater in mine, petroleum pipe line and gasser.SRB utilizes SO
4 2-to degrade some simple organism as final electron acceptor(EA), and the H existed in consumption system in metabolic process
+, produce the H of high density
2s and CO
2, thus control acidifying.At present, the SRB found reaches more than 60 genus, more than 220 and plants, by the Molecular Evolutionary Analysis of 16S rRNA gene order in database, SRB can be divided into the monoid that 4 main: Gram-negative produces the ancient bacterium of gemma sulphate reducing bacteria, sulphate-reducing thermophilic bacterium and sulphate-reducing thermophilic addicted to warm sulphate reducing bacteria, Gram-positive.But the most SRB of occurring in nature is neutrophilic, optimum pH about 6 ~ 8, and grow when pH<5 and is suppressed, and so just limits the application of sulphate reducing bacteria in control acidifying.But researchist finds that sulfate-reducing process also can occur in sour environment, this just shows that existence one class can realize the microorganism of sulfate reduction at low ph conditions, namely addicted to sour sulphate reducing bacteria (aSRB).But, all the time, the pure SRB addicted to acid (or acidproof) of separation and Culture ends in failure, until 1999, Sen & Johnson just to isolate in three strain morphological specificitys the gram-positive microorganism of relatively Desulfotomaculun genus.But up to now, SRB more than ten strains only addicted to acid (acidproof) delivered, that understands its function through genome sequencing only has 2 strains.
In the patent in the past delivered, great majority sulphate reducing bacteria are used for heavy metal-containing wastewater treatment field (CN101628773A, CN101195859A etc.), more accidental patent uses sulfate-reducing bacteria to be used for Mining Wasteland acidifying and controls to repair, but these technical schemes often also exist, and the Bacterial community of use is failed to understand, bacterial strain not acid resistance, repairing effect instability, mostly rests on laboratory stage, can not adapt to the series of problems such as actual extensive reparation very well.Such as, patent CN101037268A discloses " a kind of method of restoring mine entironment ", acidifying sulfate-reducing bacteria being used for Tailings Dam is administered, its technical scheme is the mine tailing producing in mine development, barren rock, metallurgical slag, beneficiation wastewater, smelting wastewater, acidic mine water and Ore stockpile leaching water etc. focus in Tailings Dam, the organism simultaneously adding mud and can be degraded by microorganisms, an artificial construction anaerobic environment in Tailings Dam, under the effect of microorganism and sulphate reducing bacteria, produce sulfonium ion and make the pH value of water in Tailings Dam increase, the various heavy metal ion of sulfonium ion precipitation solidification is moved to prevent it.But the sulphate reducing bacteria that this method uses derives from the Natural Selection of mud, have that said Bacterial community is above not clear, bacterial strain not acid resistance, repairing effect instability problem; Patent inventor's yet only related experiment at lab simulation, is not amplified in actual Tailings Dam and is verified, cannot be ensured the effect in practical application; Meanwhile, the method needs to pile up process, can not be suitable for the Tailings Dam having closed storehouse well, also easily cause Tailings Dam structural instability, easily have the risk of rubble flow; Finally, this technical scheme also cannot realize the effect of zoology green-recovery, land reclamation, the achievement that acidifying control obtains before follow-up phase cannot be consolidated.
Summary of the invention
The object of the invention is to overcome prior art high cost, the Bacterial community used is not clear, bacterial strain not acid resistance, repairing effect instability, mostly rest on laboratory stage, actual extensive reparation can not be adapted to very well, can not from shortcomings such as Sources controlling mine soil acidifyings, provide a kind of novel addicted to sour sulfate reduction bacteria strain.
For solving the problems of the technologies described above, the present invention is realized by following technical scheme.
The invention discloses a kind of addicted to sour sulfate reduction bacteria strain FKB, for desulfurization bends spore bacterium Desulfospornosinus sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 27th, 2014, deposit number is: CGMCC No.10072.
The invention also discloses a kind of making method containing the described liquid microbial inoculum addicted to sour sulfate reduction bacteria strain FKB, comprise the following steps:
1) FKB strain inoculation is arrived
enrichment mediumin cultivate, nitrogen is positioned over 30 DEG C of incubator quiescent culture and becomes prepared Chinese ink look to nutrient solution from clarification after blowing 20min;
2) be seeded in 10L seed fermentation tank by cultured bacterium liquid by 5% inoculum size, logical nitrogen 20min, 30 DEG C are cultured to nutrient solution and become prepared Chinese ink look from clarification;
3) by the fermented liquid in seed fermentation tank by 15% inoculum size be seeded in fermentor tank, logical nitrogen 30min, fermentation condition is:, ferment to nutrient solution and become prepared Chinese ink look;
4), after having fermented, liquid bacterial agent is made in fermented liquid pack, ensures bagging process not introducing air.
Further, described step 1) in enrichment culture based formulas be: (NH
4)
2sO
40.45g, KCl0.05g, MgSO
47H
2o 0.5g, KH
2pO
40.05g, Ca (NO
3)
24H
2o 0.014g, yeast extract 0.2g, glycerine 0.92g, distilled water 1000mL, use 1M H
2sO
4regulate pH to 3.5,121 DEG C of autoclaving 20min, after cooling, add the FeSO through filtration sterilization again
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g.
Further, described step 2) in culture medium prescription (w/v) be: cane molasses 5%, yeast leach liquor 2%, glucose 0.5%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o 0.14%, FeSO
47H
2o 5%, xitix 0.5%, pH 5.2.
The making method of liquid microbial inoculum according to claim 2, is characterized in that, described step 3) described in fermentation condition be: 30 DEG C, do not stir, pH 5.2, tank pressure 0.05 ~ 0.07Mpa; Fermentor cultivation based formulas (w/v) is: cane molasses 10%, yeast leach liquor 2%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o 0.14%, FeSO
47H
2o 6%, xitix 0.5%, pH 5.2.
Content of the present invention also comprises described addicted to sour sulfate reduction bacteria strain FKB, is controlling the application in soil acidification.
Further, described in be applied as: described is made liquid microbial inoculum addicted to sour sulfate reduction bacteria strain FKB, after soil lime, chicken manure, phosphate fertilizer etc. being carried out to simple improvement and plant stability technical tie-up use.Its concrete steps are:
1) at soil surface excavation bar ditch;
2) add lime in order, chicken manure, phosphate fertilizer improves soil matrix, described phosphate fertilizer addition is 30g/m2, manually evenly sows to surface, both sides earthen backfill, retains the degree of depth of bar ditch about 20cm, keeps 1 month;
3) add in bar ditch according to the FKB microbial inoculum consumption of every mu of 15kg, with both sides earthen backfill slightly compacting, act on 14 days;
4) employing fish scale pit, bar ditch gap plum blossom point arranges kind of a plant hole, and planting plants nutrient bag seedling, sows plant seed and soil seed bank, soil seed pool in addition, cladding thickness about 4cm straw, ensures good ventilation environment;
5) foster: need foster three times altogether between planting season, main contents comprise inspection surviving rate for the first time, ridging, and carry out after-culture; Second time content comprises loosens the soil, expands cave, adds lime, executes NPK composite fertilizer, finds dead strain after-culture immediately; Third time fosters, and main contents comprise and add lime, loosen the soil, expand cave, ridging, desinsection, and often spread manuer in holes the NPK composite fertilizer 100g that topdresses, and cooperation is loosened the soil, earthed up, and in fertilizer shallow embedding soil, if do not rain for a long time, suitably waters.
Further, described step 2) in, lime used is white lime; Chicken manure used is band husk chicken manure, carries out sterilization before using; Phosphate fertilizer used is calcium superphosphate.
Of the present invention addicted to sour sulfate-reducing bacteria strain FKB, deposit number CGMCC No.10072, by separation and purification in the mud sample of all mouth lead-zinc ore tailings bottoms of the reservior, drawn by the comparison of 16S rRNA gene order, the Desulfosporosinus that it belongs under Firmicutes door belongs to.By adopting conventional gramstaining, spore staining and scanning electron microscopic observation, result shows that FKB colony edge is irregular, shaft-like, belong to sporiferous Gram-negative bacteria, and size is 3.0 ~ 6.5 × 0.4 ~ 0.7 μm.Bio-chemical characteristics subsequently detects and shows, the physio-biochemical characteristics of FKB are as follows: growth temperature range is 10 ~ 40 DEG C, and optimum growth temperature is 30 ~ 35 DEG C; The pH scope of growth is 3.6 ~ 6.0, and the pH of optimum growth is 5.2; The NaCl concentration range of growth is 0 ~ 1.8% (w/v), and the NaCl concentration of optimum growth is 0.6%.FKB can utilize vitriol, sulphite, thiosulphate and elemental sulfur as unique sulphur source and electron acceptor(EA), and reduction reaction or disproportionation reaction generation H occur
2s generate energy are for thalli growth.FKB can also utilize arsenate as electron acceptor(EA), As (V) is reduced to As (III), and As (III) can be discharged cell under specific transporters effect, thus plays the effect reducing cellular toxicity.Nitrate also can as electron acceptor(EA) for FKB provides energy needed for growth.We thank further by the sulfo-of genome sequencing technology to this bacterium and control relevant critical function to a series of acidifyings such as the stress response of the environment such as low pH, high heavy metal ion mechanism and are studied and understand, whereby, we can use it for the acidifying control of Mining Wasteland soil better.
Described addicted to sour sulfate-reducing bacteria strain FKB, by following steps screening and separating gained:
Bed mud sample is gathered from Tailings Dam, after sample collecting, first special enrichment medium is adopted to carry out enrichment culture, the substratum of enrichment culture aSRB cultivates sulphate reducing bacteria substratum used with reference to Sen and Johnson (1999), and do corresponding improvement according to practical situation, enrichment process is as follows: get the anaerobism bottle that 1g sample to be separated is placed in the 150mL of sterilizing in advance, add 120mL enrichment medium, with high-purity nitrogen stripping 20min to remove the O of substratum
2, after air-blowing, culture is placed in 30 DEG C of constant incubator lucifuge quiescent culture, until nutrient solution becomes prepared Chinese ink look from clarification, the nutrient solution getting 5% is forwarded in fresh enrichment medium, turns training 10 times to remove most heterotrophic organism;
Sample is after enrichment culture, adopt improvement isolation medium carry out separation and purification, screening make new advances addicted to sour sulfate reduction bacteria strain, separation and purification process is as follows: configuration isolation medium, be incubated at about 50 DEG C after sterilizing, the bacterium liquid after enrichment culture is diluted to 10
-2~ 10
-6the bacteria suspension of concentration, get the different dilution bacterium liquid of 50 μ L respectively in culture dish, pour isolation medium immediately into and rock culture dish gently, while hot bacterium liquid is mixed with substratum, identical substratum is poured again into after agar solidification, then culture dish is put into the crisper being added with anaerobic gas generation bag (Mitsubishi) and carry out Anaerobic culturel in 30 DEG C, about about 2 weeks, the bacterium colony of appearance black is grown in culture dish, picking list bacterium colony proceeds to Anaerobic culturel in liquid nutrient medium, repeat above-mentioned steps until the colonial morphology grown in culture dish is consistent, can identify being separated the SRB obtained, preserve.
Described method for screening and separating, its culture medium prescription used is:
Enrichment medium: (NH
4)
2sO
40.45g, KCl 0.05g, MgSO
47H
2o 0.5g, KH
2pO
40.05g, Ca (NO
3)
24H
2o 0.014g, yeast extract 0.2g, glycerine 0.92g, distilled water 1000mL, use 1M H
2sO
4regulate pH to 3.5,121 DEG C of autoclaving 20min, after cooling, add the FeSO through filtration sterilization again
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g;
Isolation medium: take corresponding medicine by enrichment culture based formulas, is dissolved in 700mL distilled water, uses 1M H
2sO
4regulate pH to 3.5; Separately taking 1g agar is dissolved in 300mL distilled water, respectively at 121 DEG C of sterilizing 20min, is cooled to about 50 DEG C, is mixed by two portions substratum, then add the FeSO of filtration sterilization
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g.
Compared with prior art, the present invention has the following advantages:
1, the present invention filter out addicted to sour sulfate-reducing bacteria strain FKB bacterial strain, it belongs to addicted to sour sulfate reduction bacteria strain, the isolated aSRBs in the current whole world only more than ten strains, not only overcome the uncertainty of passing use microorganism species, and oxyphilous feature also allows this bacterial strain more can adapt to extreme acid condition, breeding is faster, metabolism is more prosperous, better effects if; Meanwhile, we have done genome sequencing to FKB, have understood its metabolism and function in depth, and especially in the machine-processed these two aspects that sulfate reduction and anti-pH are coerced, this greatly can help us to cultivate better and utilize this bacterial strain to control for acidifying.
2, control soil acidification technology of the present invention, sulfate-reducing bacteria is adopted to carry out acidifying control, the generation of acidifying is suppressed from Mining Wasteland acidifying root, simultaneously after soil lime, chicken manure, phosphate fertilizer etc. being carried out to simple improvement and plant stability technical tie-up use, avoid the potential threat that after-souring occurs similar technique after repair, avoid again repairing required a large amount of manpower and materials and the wasting of resources, also can reach the object of zoology green-recovery, mine reclamation simultaneously.
3, control soil acidification technology of the present invention has safety and environmental protection, eco-friendly feature, without the need to carrying out big area transformation to original landforms, also without the need to carrying out large-area soil moved in improve the original, avoids the destruction of fetching earth to periphery mountain forest ecotope.All raw material and whole repair process all can not bring secondary pollution to environment.
4, control soil acidification technology of the present invention has feature with low cost, simple to operate, and in YONGPING COPPER DEPOSIT refuse dump, Ores At Chengmenshan Copper Mine Tailings Dam carried out big area practical application and achieved good effect, acidification phenomenon is obviously controlled, vegetation coverage, all more than 90%, is applicable to large-area promoting the use of.
In order to understand better and implement, describe the present invention in detail below in conjunction with accompanying drawing.
Accompanying drawing explanation
Fig. 1 is the gramstaining addicted to sour sulfate-reducing bacteria strain FKB bacterial strain of the present invention (1,000 times of opticmicroscope) and scanning electron microscopic observation aspect graph.
Of the present invention addicted to sour sulfate reduction bacteria strain FKB, for desulfurization bends spore bacterium Desulfospornosinus sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 27th, 2014, deposit number is: CGMCC No.10072.
Embodiment
The qualification of embodiment 1FKB strains separation and biological function explore
1) bacterial screening
From the bed mud sample of all mouth Pb-Zn deposits No. 1 Tailings Dam edge collecting, collect with aseptic 50mL centrifuge tube, be then placed in ice bath and transport laboratory back, be kept at 4 DEG C of refrigerators immediately.
After sample collecting, first adopt special enrichment medium to carry out enrichment culture, object allows the sulfate reduction flora originally not occupying superiority be able to amount reproduction, and remove most heterotrophic organism.The substratum of enrichment culture aSRB cultivates sulphate reducing bacteria substratum used with reference to Sen and Johnson (1999), and does corresponding improvement according to practical situation.The composition of enrichment medium is: (NH
4)
2sO
40.45g; KCl0.05g; MgSO
47H
2o 0.5g; KH
2pO
40.05g; Ca (NO
3)
24H
2o 0.014g; Yeast extract 0.2g; Glycerine 0.92g, distilled water 1000mL, use 1M H
2sO
4regulate pH to 3.5,121 DEG C of autoclaving 20min, after cooling, add the FeSO through filtration sterilization again
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g.Enrichment process is as follows: get the anaerobism bottle that 1g sample to be separated is placed in the 150mL of sterilizing in advance, add 120mL enrichment medium, with high-purity nitrogen stripping 20min to remove the O of substratum
2.After air-blowing, culture is placed in 30 DEG C of constant incubator lucifuge quiescent culture, until nutrient solution becomes prepared Chinese ink look from clarification, shows sulphate reducing bacteria amount reproduction.The nutrient solution getting 5% is forwarded in fresh enrichment medium, turns training 10 times to remove most heterotrophic organism.
Sample after enrichment culture, adopt improvement isolation medium carry out separation and purification, screening make new advances addicted to sour sulfate reduction bacteria strain.The formula of isolation medium is as follows: take corresponding medicine by enrichment culture based formulas, is dissolved in 700mL distilled water, uses 1M H
2sO
4regulate pH to 3.5; Separately taking 1g agar is dissolved in 300mL distilled water, respectively at 121 DEG C of sterilizing 20min, is cooled to about 50 DEG C, is mixed by two portions substratum, then add the FeSO of filtration sterilization
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g.Separation and purification process is as follows: configure isolation medium according to the method described above, be incubated at about 50 DEG C after sterilizing, bacterium liquid after enrichment culture is diluted to the bacteria suspension of 10-2 ~ 10-6 concentration, get the different dilution bacterium liquid of 50 μ L respectively in culture dish, pour isolation medium immediately into and rock culture dish gently, while hot bacterium liquid is mixed with substratum, after agar solidification, pour identical substratum again into, then culture dish is put into the crisper being added with anaerobic gas generation bag (Mitsubishi) and carry out Anaerobic culturel in 30 DEG C.About about 2 weeks, grow the bacterium colony of appearance black in culture dish, picking list bacterium colony proceeds to Anaerobic culturel in liquid nutrient medium.Repeat above-mentioned steps until the colonial morphology grown in culture dish is consistent, can identify, preserve being separated the SRB obtained.
2) strain identification and biological function explore
Molecular biology identification: be separated after obtaining FKB, carry out extracting genome DNA to it, adopts 27F/1492R amplification 16s rRNA gene order, delivers to Hua Da gene and check order, obtain its sequence information after pcr product purification.Utilize NCBI BLAST that the 16S rRNA sequence information of measured FKB bacterial strain and GenBank database sequence are carried out tetraploid rice, result shows that FKB belongs to Clostridiales order, Peptococcaceae section, Desulfosporosinus belong to (microorganism in this genus all has sulfate reduction function).
Morphological features qualification and physio-biochemical characteristics: the form of thalline adopts conventional gramstaining, spore staining and scanning electron microscopic observation.Result is: FKB colony edge is irregular, shaft-like, belong to sporiferous Gram-negative bacteria, and size 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, and optimum growth temperature is 30 ~ 35 DEG C; The pH scope of growth is 3.6 ~ 6.0, and the pH of optimum growth is 5.2; The NaCl concentration range of growth is 0 ~ 1.8% (w/v), and the NaCl concentration of optimum growth is 0.6%.FKB can utilize vitriol, sulphite, thiosulphate and elemental sulfur as unique sulphur source and electron acceptor(EA), and reduction reaction or disproportionation reaction generation H occur
2s generate energy are for thalli growth.FKB can also utilize arsenate as electron acceptor(EA), As (V) is reduced to As (III), and As (III) can be discharged cell under specific transporters effect, thus plays the effect reducing cellular toxicity.Nitrate also can as electron acceptor(EA) for FKB provides energy needed for growth.
Full-length genome measures: in order to more clearly understand the acidifying controlling mechanism of FKB, we have carried out genome sequencing to it.First extract the genomic dna of FKB, be then broken into by Covaris sonicator the fragment that length is 500 and 800bp at random, through end reparation, add sequence measuring joints, preparation that purifying, pcr amplification complete library.The library built utilize IlluminaMiseq PE 250 sequenator to its carry out two generation high-flux sequence.Sequencing data is through quality control, sequence assembly, then the draft genome of splicing gained is carried out predictive genes by Genemark, and use BLASTx that functional annotation is carried out in the open reading frame obtained (ORF) and the functional database comparison such as NCBI-nr (Non redundant), KEGG, egg-NOG; Simultaneously, predicted gene is uploaded KAAS and carry out functional annotation, and by building its metabolic pathway to the basal metabolism of each bacterial strain metabolism such as () C, N, P, S and resistance (comprising heavy metal, pH resistance and oxidative stress etc.) response mechanism.
The Genome Size of FKB is 5.1Mbp, is made up of 40 scaffold, and G+C ratio is 41.8%; Its 16s rDNA sequence is as shown in sequence table Seq ID No.1.From 16S rRNA phylogeny, to cls gene group bacterial strain Desulfosporosinus acidiphilus SJ4 is comparatively similar, similarity is 96%.In FKB, we detect coding catalysis vitriol, sulphite and the full gene needed for thiosulfuric acid salt metabolism.First, intracellular SO is entered
4 2aPS is formed through vitriol adenosyl transferase (sat, sulfate adenylyltransferase) catalysis; Then, SO is generated by adenosine sulfate reduction enzyme (adenylylsulfate reductase) the catalysis APS of aprAB genes encoding
3 2-, eventually through sulfate reduction enzyme (dissimilatory sulphate reductase) the catalysis SO of dsrAB genes encoding alienation
3 2-generate H
2s.Further, the phsA gene encodes thiosulphate reduction enzyme (thiosulfate reductase) detected in FKB genome, this enzyme energy catalysis thiosulphate reduction generates H
2s.On heavy metal resistance, FKB goes reply to coerce by three kinds of modes: one be the ionic pump that participates in of ATP enzyme to get rid of harmful heavy metal ions, two is that three is oxidizing reactions of heavy metal cation by forming complex compound to reduce heavy metal toxicity.In addition, coerce for low pH, genome sequencing result display FKB is responded by following four kinds of modes: 1, pass through Na
+/ H
+aTPase, K
+/ H
+the exchange that ATPase realizes proton is discharged; 2, a series of tenuigenin buffer molecule absorbs the proton penetrating into cell; 3, catalysis ornithine, Methionin and arginine decarboxylation consume proton; 4, proton removed by multiple organic acid of degrading.The existence of and multiple pH stress response mechanism machine-processed just because of above sulfate reduction mechanism, heavy metal resistance, FKB just has survival under low pH, high density heavy metal ion condition and has sulphate reducing consumption H
+control the function of acidifying.Whereby, we can use it for the acidifying control of Mining Wasteland soil better on the basis having understood FKB life habit and function in detail.
Prepared by embodiment 2FKB liquid bacterial agent
1) cultivate in FKB strain inoculation to enrichment medium, use the blue cover glass bottle of 100mL, need nitrogen to blow 20min after inoculation and remove air, be then positioned over 30 DEG C of incubator quiescent culture and become prepared Chinese ink look to nutrient solution from clarification;
2) cultured bacterium liquid is seeded in 10L seeding tank by 5% inoculum size, logical nitrogen 20min, 30 DEG C are cultured to nutrient solution and become prepared Chinese ink look from clarification, the canned liquid measure of seed fermentation is 70%, culture medium prescription (w/v) is: cane molasses 5%, yeast leach liquor 2%, glucose 0.5%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o 0.14%, FeSO
47H
2o5%, xitix 0.5%, pH 5.2;
3) by the fermented liquid in seed fermentation tank by 15% inoculum size be seeded in fermentor tank, logical nitrogen 30min, fermentation condition is: 30 DEG C, do not stir, pH 5.2, tank pressure 0.05 ~ 0.07Mpa, fermentation to nutrient solution becomes prepared Chinese ink look, fermentor cultivation based formulas (w/v) is: cane molasses 10%, yeast leach liquor 2%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o0.14%, FeSO
47H
2o 6%, xitix 0.5%, pH 5.2; Undeclared part is undertaken by fermentation general operation;
4), after having fermented, liquid bacterial agent is made in fermented liquid pack, ensures bagging process not introducing air.
Embodiment 3FKB is used for refuse dump acidifying and controls
Place is refuse dump, YONGPING COPPER DEPOSIT south, Jiangxi, and area is 16800 square metres, is divided into top and side slope two large regions, side slope 10000m
2, top 6800m
2.This region has following Typical Representative meaning:
(1) side slope is steep, drop is large: according to terrain map of survey and drawing, and this region cope level 273.3m, toe elevation 185.5m, the discrepancy in elevation reaches 87.8m, and grade of side slope is between 1:1 ~ 1:1.25.Substantially the situation that landform side slope is steep, drop is large of refuse dump can be represented.
(2) wash away seriously, erratic boulder is in heaps: due to for many years and wash away, it is erratic boulder substantially that this regional slope and toe account for the total area more than 60%, without soil, for the condition that the survives extreme difference (scene has no any plant-growth) of plant, belong to the region that Revegetation difficulty in refuse dump is relatively large.
(3) side slope soil property situation is poor: as can be seen from field condition, although still some soil near this region intermediate platform, rarely seen plant-growth, during scene has acidification reaction just acutely to carry out as seen, produces outside hot gas and emit.
After the prepartion of land of trial plot, carry out sampling analysis to whole trial plot soil, sampling depth is 0 ~ 20cm, obtains pedotheque 96 altogether.Analytical results shows: this trial plot belongs to strongly-acid, high Acidification potential, there is the soil of a large amount of free acid ion simultaneously; And also there is very serious cupric ion toxicity hazard in trial plot, there is lighter Toxicity of Lead subregion; Soil nutrient is very deficient, can not meet plant germination far away and grows necessary macronutrient.Concrete analysis data see the following form:
Note: NAG unit is kg H
2sO
4/ t, heavy metal index unit is ppm, and nutritive element index unit is g/kg.
According to above soil sample investigation result, determine that chicken manure addition is 12.5kg/m
2, white lime addition is 7kg/m
2, phosphate fertilizer addition is 30g/m
2; The laboratory experiment screening done by the refuse dump soil of delivery in early stage and examine on the spot, determine that plant growing scheme is as follows: plantation Caulis Miscanthis floriduli, Pinus massoniana Lamb, locust tree nutrient bag seedling; The seed such as live Festuca Arundinacea, Bermuda grass, paspalum notatum, paulownia, sesbania, giant knotweed, lettuce fiber crops; Separately add soil seed bank, soil seed pool, soil seed bank, soil seed pool takes from local discarded farmland.Concrete operation step is:
1) at soil surface excavation bar ditch;
2) add lime in order, chicken manure, phosphate fertilizer improves soil matrix, described phosphate fertilizer addition is 30g/m2, manually evenly sows to surface, both sides earthen backfill, retains the degree of depth of bar ditch about 20cm, keeps 1 month;
3) add in bar ditch according to the FKB microbial inoculum consumption of every mu of 15kg, with both sides earthen backfill slightly compacting, act on 14 days;
4) employing fish scale pit, bar ditch gap plum blossom point arranges kind of a plant hole, and planting plants nutrient bag seedling, sows plant seed and soil seed bank, soil seed pool in addition, cladding thickness about 4cm straw, ensures good ventilation environment;
5) foster: need foster three times altogether between planting season, main contents comprise inspection surviving rate for the first time, ridging, and carry out after-culture; Second time content comprises loosens the soil, expands cave, adds lime, executes NPK composite fertilizer, finds dead strain after-culture immediately; Third time fosters, and main contents comprise and add lime, loosen the soil, expand cave, ridging, desinsection, and often spread manuer in holes the NPK composite fertilizer 100g that topdresses, and cooperation is loosened the soil, earthed up, and in fertilizer shallow embedding soil, if do not rain for a long time, suitably waters.
Trial plot is from construction of marching into the arena at the beginning of 2013 11 months, complete prepartion of land, base material improvement before year and add microbial inoculum work, after the beginning of spring, weather has got warm again after a cold spell plant growing work, on March 20th, 2014 the complete items of cost whole construction workings, what transfer the later stage to fosters work.On June 9th, 2014, We conducted trial plot situation of repairing and investigate and botanizing work.Find through field trip, the acidifying situation of trial plot is greatly improved, do not see ongoing acidification reaction, Soil nutritional condition significantly improves, multiple short herbaceous plant occupies superiority, as paspalum notatum, Chinese silvergrass, Bermuda grass etc., soil erosion situation originally obtains effective containment.Botanizing result display trial plot has plant variety 47 kinds, and contain draft, shrub, arbor, overall coverage reaches 90%, and ensemble average height is 50cm, and riotous growth, grows fine.On September 11 the same year, We conducted second time site inspection and botanizing work.Result shows, trial plot acidifying situation is clearly better, the average cover degree of vegetation is 96%, whole height is 160cm, the overall growing state of plant is good, the short draft originally occupying superiority by some tall and big draft, shrub or arbor replace, as Caulis Miscanthis floriduli, pale persicaria, ramie, sesbania, Formosan paulownia (Cortex seu Radix Paulowniae kawakamii) etc., under soil property had and comparatively significantly changed, in water-retentivity, fertility, stability etc., had large increase.The ecotope of whole trial plot is obviously promoted.
Embodiment 4FKB is used for Tailings Dam acidifying and controls
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 will be stopped using the end of the year, and so far by 6 ~ 7 years, tailings drying and consolidating in Tailings Dam, check surface, has certain bearing capacity of foundation soil, and Tailings Dam reservoir area major part is withered, and during rainy season, in storehouse, local can ponding.According to reconnaissance trip, sample plot surface is smooth, and surface is powder white sand shape, and lower floor is closely knit adhesion then, and the phenomenon that hardens is very serious; Whole sample plot scope is without any plant-growth, and surface temperature is high, lack of water, and water retention is poor; Soil layer is soft, and people tramples and can leave darker vestige, is extremely unfavorable for the operation of heavy construction apparatus.Early stage has carried out sampling survey work, and sampling depth is 0 ~ 20cm, and gross sample number is 50, and Testing index comprises acidifying index, heavy metal index and nutritive element index three bulk.Sample analysis result shows that this Tailings Dam trial plot soil acidification is very serious, and pH is low reaches 2.56, and there is higher Acidification potential, and NAG reaches 21.5kg H
2sO
4/ t, cupric ion is poisoned serious, and there is a serious shortage in the supply for plant-growth desired nutritional element.Concrete analysis the results are shown in following table:
Note: NAG unit is kg H
2sO
4/ t, heavy metal index unit is ppm, and nutritive element index unit is g/kg.
According to above soil sample investigation result, determine that chicken manure addition is 15kg/m
2, white lime addition is 10kg/m
2, phosphate fertilizer addition is 30g/m
2; According to technical scheme operation described in embodiment 3.The laboratory experiment screening done by the Tailings Dam soil of delivery in early stage and examine on the spot, determine that plant growing scheme is as follows: plantation Herba panici repentics, ramie, Siberian cocklebur, locust tree nutrient bag seedling; The seed such as live Festuca Arundinacea, Bermuda grass, paspalum notatum, sesbania, pale persicaria; Separately add soil seed bank, soil seed pool, soil seed bank, soil seed pool takes from local discarded meadow.
Project was marched into the arena on January 6th, 2014, and on February 5th, 2014 completes the construction working of experimental project substantially, turned to foster work after rear construction simultaneously.On June 28th, 2014, we have carried out botanizing and soil sample analysis to trial plot.Botanizing result shows that trial plot has plant 26 kinds, begin to take shape the growth situation that various plants coupling is long mutually, the average plant height of plant reaches 50cm, the root system degree of depth at more than 10cm, the average plant height 20cm of leguminous plants, the root system degree of depth are at more than 20cm, leguminous plants has defined good root nodule bacterium, enhances plant nitrogen fixing capacity.Vegetation cover degree reaches more than 85%, each kind root system is crisscross, complementary, the root system network on formation control tailings earth's surface, microorganism and root system of plant acting in conjunction in plant substrates, the foundation of vegetation impels tailings to become native trend obviously to accelerate, and realizes the target controlling acidifying, zoology green-recovery.Soil sample analytical results shows, after reparation about 5 months, sample plot pH rises to 7.90, and strongly-acid is effectively improved; NAG-pH is 3.38, and potential product acid is controlled effectively; EC value is down to 0.95ms/cm by 2.34ms/cm, and in soil, a large amount of free murder by poisoning ion existed greatly reduces, and the acidifying situation of whole sample plot is effectively improved.This project is at present by checking and accepting by Jiangxi Copper Co., Ltd. smoothly.
Claims (9)
1. addicted to a sour sulfate reduction bacteria strain FKB, it is characterized in that: this bacterial strain belongs to desulfurization and bends spore bacterium (Desulfospornosinus sp.), and its deposit number is: CGMCC No.10072.
2. the making method containing the liquid microbial inoculum addicted to sour sulfate reduction bacteria strain FKB according to claim 1, comprises the following steps:
1) FKB strain inoculation is arrived
enrichment mediumin cultivate, anaerobism 30 DEG C of quiescent culture become prepared Chinese ink look to nutrient solution from clarification;
2) be seeded in fermentor tank by cultured bacterium liquid by 5% inoculum size, anaerobism 30 DEG C of quiescent culture become prepared Chinese ink look to nutrient solution from clarification;
3) by the fermented liquid in seed fermentation tank by 15% inoculum size be seeded in fermentor tank, anaerobism 30 DEG C of quiescent culture to nutrient solutions become prepared Chinese ink look;
4), after having fermented, liquid bacterial agent is made in fermented liquid pack, ensures bagging process not introducing air.
3. the making method of liquid microbial inoculum according to claim 2, is characterized in that: described step 1) in enrichment culture based formulas be: (NH
4)
2sO
40.45g, KCl 0.05g, MgSO
47H
2o 0.5g, KH
2pO
40.05g, Ca (NO
3)
24H
2o 0.014g, yeast extract 0.2g, glycerine 0.92g, distilled water 1000mL, use 1M H
2sO
4regulate pH to 3.5,121 DEG C of autoclaving 20min, after cooling, add the FeSO through filtration sterilization again
47H
2o 0.5g, xitix 0.02g, mercaptoethanol 1g.
4. the making method of liquid microbial inoculum according to claim 2, is characterized in that: described step 2) in culture medium prescription (w/v) be: cane molasses 5%, yeast leach liquor 2%, glucose 0.5%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o0.14%, FeSO
47H
2o 5%, xitix 0.5%, pH 5.2.
5. the making method of liquid microbial inoculum according to claim 2, is characterized in that: described step 3) described in fermentation condition be: 30 DEG C, do not stir, pH 5.2, tank pressure 0.05 ~ 0.07Mpa; Fermentor cultivation based formulas (w/v) is: cane molasses 10%, yeast leach liquor 2%, (NH
4)
2sO
44.5%, MgSO
47H
2o 5%, KCl 0.5%, KH
2pO
40.5%, Ca (NO
3)
24H
2o 0.14%, FeSO
47H
2o 6%, xitix 0.5%, pH 5.2.
6. according to claim 1 addicted to sour sulfate reduction bacteria strain FKB, controlling the application in soil acidification.
7. application according to claim 6, is characterized in that: make liquid microbial inoculum by according to claim 1 addicted to sour sulfate reduction bacteria strain FKB, after improvement is carried out to soil lime, chicken manure, phosphate fertilizer etc. and plant stability technical tie-up use.
8. application according to claim 7, its concrete steps are:
1) at soil surface excavation bar ditch;
2) add lime in order, chicken manure, phosphate fertilizer improves soil matrix, described phosphate fertilizer addition is 30g/m
2, manually evenly sow to surface, both sides earthen backfill, retain the degree of depth of bar ditch about 20cm, keep 1 month;
3) add in bar ditch according to the FKB microbial inoculum consumption of every mu of 15kg, with both sides earthen backfill slightly compacting, act on 14 days;
4) employing fish scale pit, bar ditch gap plum blossom point arranges kind of a plant hole, and planting plants nutrient bag seedling, sows plant seed and soil seed bank, soil seed pool in addition, cladding thickness about 4cm straw, ensures good ventilation environment;
5) foster: need foster three times altogether between planting season, main contents comprise inspection surviving rate for the first time, ridging, and carry out after-culture; Second time content comprises loosens the soil, expands cave, adds lime, executes NPK composite fertilizer, finds dead strain after-culture immediately; Third time fosters, and main contents comprise and add lime, loosen the soil, expand cave, ridging, desinsection, and often spread manuer in holes the NPK composite fertilizer 100g that topdresses, and cooperation is loosened the soil, earthed up, and in fertilizer shallow embedding soil, if do not rain for a long time, suitably waters.
9. application according to claim 8, is characterized in that, described step 2) in, lime used is white lime; Chicken manure used is band husk chicken manure, carries out sterilization before using; Phosphate fertilizer used is calcium superphosphate.
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