CN101434909A - Penicillium, as well as preparation method and application - Google Patents
Penicillium, as well as preparation method and application Download PDFInfo
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Abstract
The invention discloses a Penicillium, a preparation method and applications thereof, Penicillium fungus PSM11-5 is separated from a vanadium ore sample; insoluble tricalcium phosphate, sodium metavanadate, cobalt hydroxide and basic nickel carbonate are taken as indicating compounds; and a fungal strain is screened by testing the capability of decomposing the tricalcium phosphate, the sodium metavanadate, the cobalt hydroxide and the basic nickel carbonate. The Penicillium PSM11-5 is Penicillium sp.PSM11-5 CCTCCM208207. The strain is utilized for carrying out biological leaching of phosphorus and biological metallurgy, metals of phosphorus, vanadium, nickel, cobalt and the like are leached from lean ores, discarded ores, submarginal ores, difficult-to-mine ores, difficult dressing ores and refractory ores, thereby fully utilizing the mineral resources, reducing the metallurgical costs and protecting the ecological environment. The PSM11-5 is utilized for leaching the phosphorus from low-grade phosphate rock powder, a biological fertilizer is prepared to be applied to the soil, thereby leading the soil to contain higher content of soluble phosphorus which can be utilized by crops; the strain further leaches insoluble phosphorus which is deposited in the soil before, thereby reducing phosphorus fertilizer and reducing gas pollution caused by the phosphorus fertilizer and water pollution caused by the phosphorus fertilizer.
Description
Technical field
The microorganism that the present invention relates to phosphorus and metal in phosphorus and the metallic ore is leached, and more specifically relates to a kind of Penicillium notatum (Penicillium) that insolubility phosphorus and metallic ore are had Decomposition, also relates to the preparation method of this bacterium simultaneously, also relates to the purposes of this bacterium.This penicillium belongs to fungi, never in solubility phosphorus ore and metallic ore such as alum ore deposit, nickel, the cobalt ore etc. phosphorus and metal is leached, and its material that plays the leaching effect mainly is organic acids such as gluconic acid.This bacterial strain is cultivated better suited the soaking in the substratum of ore deposit of containing ground phosphate rock or metal mining powder, phosphorus and metal can be leached in the nutrient solution from mineral, so this bacterial strain can be used for the preparation of phosphobacterin or be used for microbial metallurgy in conjunction with the routine techniques of industrial hydrometallurgy.
Background technology
In very long geological age, microorganism has participated in the circulation of earth's crust chemical element.Many microorganisms can be by number of ways to the mineral effect, discovers that lawn that blue-green algae on quartzite and fungi form can destroy the crystalline structure of quartzite, forms forsterite mineral (Geomicrobiol J, 2001,18 (1): 117-132) again.The microorganism of occurring in nature can be destroyed mineral crystal structure makes wherein valuable element be converted into ion in the solution.Utilize this specific character of microorganism,, formed the biological metallurgy technology in conjunction with related process such as hydrometallurgys.Put down in writing from document, China is the country that adopts the biological metallurgy technology in the world the earliest, as far back as B.C. 2nd century, just put down in writing with the chemical action of iron from the mid-recopper of copper-bath, dump leaching is exactly common practice (the MineSci Eng of production of copper at that time, 1980,12 (3): 121-189).In Europe, biological metallurgy activity the earliest on the books be 1670 in Hispanic RioTinto ore deposit, people from pit water, reclaim the copper that bacterium leaches (Spain TransInstMin Metal, 1943,52:35-71).
The paces of world today's development are accelerated day by day, are with rapid changepl. never-ending changes and improvements, and the demand to various Mineral resources also strengthens day by day simultaneously, and the higher-grade rich ore of phosphorus ore and various metals is faced with the situation of petering out.Therefore extraction phosphorus and metal from low grade ore, complex ore, unbalanced-ore, mine tailing and trade waste are subjected to people's attention and attention more and more.Traditional pyrometallurgy and hydrometallurgy exist that the rate of recovery is low, process is complicated, the High Temperature High Pressure process need consumes lot of energy, environmental pollution is serious and can't handle weak point such as a lot of difficult, low-grade, complicated mineral deposits.And biological soak phosphorus and biological metallurgy technology be particularly suitable for lean ore, abandoned mine, unbalanced-ore and difficultly adopt, the dump leaching in difficult choosing, difficult smelting ore deposit and leaching on the spot, and have outstanding advantages such as process is simple, cost is low, energy consumption is low, environmental pollution is little.Utilize this technology not only can from low grade ore, extract valuable metals such as phosphorus and copper, gold, uranium, nickel, cobalt simply, efficiently, (the Geomicrobiol J that poisons that can also be used for mine reclamation, waste metal treatment and remove sewage sludge, 2004,21 (3): 135-144).
Phosphorus is important chemical material, also is the necessary element of crop growth, and industrial phosphorus must extract from phosphorus ore in a large number, is used to produce phosphate fertilizer, the multiple fertilizer of phosphorus and meticulous phosphorus chemical product.Because its utility value enjoys attention in industry such as agricultural, industry, medicine and civil area, phosphate rock resource progressively becomes a kind of strategic resource in the world.Yet phosphate rock resource is progressively moving towards exhausted as a kind of Nonrenewable resources.States such as the U.S., India and Russia have started emergent research work, and the Rock Phosphate (72Min BPL) reserves that China lists national statistics in are 16,800,000,000 t.The characteristics of China's phosphate rock resource are that resource reserve is bigger, distribute to concentrate; Middle low grade ore is many, and rich ore is few; Collophanite is many, adopts, selects difficulty big; Deposit type is based on the deposition phosphorite.The mining type of China's phosphorus ore is mainly underground mining and strip mining transformation two big classes at present: the mining methods of underground mining are mainly open-stope method and avalanche method; Opencast ore-dressing technique mainly is a direct flotation technology, the roasting digestion, scrubbing-desliming, anti--beneficiation enrichment technologies such as (just-anti-) flotation, dense medium just of deposition phosphorite have also been developed in recent years in succession, in these technology, direct flotation, reverse flotation, scrubbing-desliming, dense medium separation are successfully used.But the development and use of the present phosphate rock resource of China exist obviously not enough, mainly show: the phosphate rock resource extensive management, and exploitation losses are serious; Production technique falls behind, and the destruction Of resources and waste are serious; Though technique of preparing has breakthrough, 70% of expense height, particularly China's phosphorus ore be in low-grade collophanite, mineral grain is thin, embedding cloth is tight, detrimental impurity is more, adopt above-mentioned technology concentration ratio the difficulty; Environment is caused expendable pollution.Along with the outstanding problem that exists in the increasing of phosphorus ore mining inetesity and the recovery process, the work-ing life of limited non-renewable phosphate rock resource is shorter and shorter, quickened the depleted speed of Chinese phosphor resource, maximum hidden danger is that prestige is being assisted in the country, the grain security in long term.Therefore it is extremely urgent to study beneficiation new process a kind of Sustainable development, comprehensive development and utilization.
Utilize microorganism can decompose this characteristic of insolubility phosphorus, make it be widely used in industrial and agricultural production, all be devoted at present both at home and abroad the separation screening of the high new bacterial strain of microorganism of leaching efficiency and the research of existing strain improvement aspect.Aspect the separation of phosphate solubilizing microorganism bacterial strain, screening, document " the phosphorus decomposing ability of two strain phosphorus decomposing fungies and the preliminary study of phosphorus decomposing mechanism thereof (microbiology circular; 2006; 33 (5): 22-26) ", " separation of the different phosphate solubilizing microorganisms of two strains and characteristic thereof (science of soil and manure; 2008; 24 (3): 239-242) " wait the separation to China's phosphate solubilizing microorganism, identify that grade done research.Be applied to agriculture aspect, " different phosphorus solution bacteria communities in calcareous soil to the influence of yield of rape and quality (Agricultural University Of Shanxi's journal; 2006; 26 (2): 149-151) ", " different phosphorus solution bacteria communities are to the research (crop magazine; 2006; 173-75) of pea growth and yield effect ", " screening that phosphorus decomposing please mould and to the production-increasing function of farm crop and herbage (Shandong science, 2005,18 (3): 78-84) " etc. phosphate solubilizing bacteria has been done research to the raising crop yield.Aspect using and leaching ground phosphate rock, " phosphate solubilizing bacteria separate and to Inner Mongol cloth dragon picture low-grade phosphate ore utilization research (earth journal; 2007; 28 (4): 377-381) ", " characteristic of 4 bamboo phosphate-solubilizing bacterias and fungi dissolved phosphorus breeze (microbiology circular; 2002; 29 (6): 24-28) ", " a strain aspergillus Aspergillus 2TCiF2 dissolved phosphorus breeze dynamically (China Agricultural University's journal; 2003; 8 (3): 43-46) ", " a strain Arthrobacter dissolved phosphorus breeze dynamically (JOURNAL OF MICROBIOLOGY; 2003,23 (5): 12-17) " etc. done preliminary research to the microbiological degradation phosphorus ore.Aspect the mechanism of microbiological deterioration insolubility phosphorus, " the soil phosphate solubilizing microorganism mechanism of action and phosphate solubilizing bacterial manure to the influence of plant growth (Anhui agricultural sciences; 2008; 36 (14): 5948-5950) ", " progress of microorganism phosphorus decomposing mechanism (Shandong agricultural sciences; 2008,288-91) " etc. have been made preliminary study to the mechanism of action.
Nickel, cobalt etc. are crucial non-ferrous metal raw materials, have premium properties, have become the indispensable metal of modernized system of the modern aircraft industry of development, national defense industry and the human high-level material and cultural life of foundation.All the time, utilizing heterotrophic microorganism biological leaching nickel, cobalt from the low ore grade nickel cobalt ore all is the focus of research.McKenzie, Denys etc. (1987) adopt the Acetate Film electrophoretic technique to study various organic acids and fungi biological nickel, cobalt mechanism of leaching from low-grade laterite type nickel cobalt ore first.(FemsMicrobiol Rev such as Alibhai and Dudeney, 1993,11 (1-3): 87-96) from certain low-grade laterite type nickel cobalt ore, leached nickel, the cobalt of 50-60%, and thought that its selectivity in mineral leaches nickel, cobalt is a reason of having participated in leaching process owing to microorganism itself directly with a penicillium biology (can tolerate the nickel of 6400Pppm high density).(Hydrometallurgy, 1993,32 (3): 373-379) obtained 85% and 92% nickel leaching yield respectively such as Sukla and Panchanadikar with bacillus circulans (Bacillus.circulans) and aspergillus niger (Aspergillus niger).Sukla and Panchanadikar (W J Microbiol ﹠amp; Biotech, 1993,9 (2): 255-257) obtained 90% and 34% nickel, cobalt leaching yield in 20 days respectively with the aspergillus tubigensis leaching.Tzeferis (Inter JMine Proc, 1994,42 (3-4): 267-283) adopt cultivation liquid to regulate the back 95 ℃ of two-step approachs of pH value and leach the nickel leaching yield that can reach 70-72.(Mine Eng such as Valix and Tang, 2001,14 (5): 499-505) studied penicillium funiculosum in the fungi leaching process (Penicillium funiculosum), smelly aspergillus (Aspergillus foetidus), the metal ion tolerance of letter mould (Penicillium simplicissimum), and leaching yield (Mine Eng, 2001,14 (2): 197-203) of 36wt% nickel, 54wt% cobalt, 0.76wt% iron have been obtained respectively from certain low-grade laterite type nickel cobalt ore with these bacterial strains.(MineEng such as Le and Tang, 2006,19 (12): 1259-1265) successful cultivation one strain many metal resistance that can tolerate the many metal ions of high density (Al, Co, Cr, Cu, Fe, Mg, Mn, Ni, Zn) soak the smelly aspergillus in ore deposit (Aspergillusfoetidus), seldom can in 12 days, from low-grade laterite type nickel cobalt ore, leach 28%, 31%, 16%, 30% Ni, Co, Fe, Mg respectively though this bacterial strain produces the acid amount in soaking the ore deposit process.Yet, complicacy is, studies show that, chemistry, mineralogy that validity that the non-sulfide ore microorganism is leached and economic feasibility depend on specific bacterial strain and certain minerals, the result that indivedual mineral and bacterial strain experiment draw can not simply be generalized to other microorganisms and mineral (Fems Microbiol Rev, 1997,20 (3-4): 591-604).Therefore every kind of mineral and technology must be carried out the research in early stage in the laboratory before using.Undoubtedly, carry out relevant research work in China and have huge theory value and actual application value.
Vanadium metal is mainly used in the alloying steel; Vanadium Pentoxide in FLAKES and vanadate are widely used as catalyzer; Also be used to make stained glass and pottery, and the siccative of paint and ink.The occurring in nature vanadium is difficult to be individual and exists, and main and other mineral form mineral intergrowth or complex ore.China's navajoite resource mainly contains the bone coal ore deposit and clay pit is formed, and extraction value and reserves are based on bone coal.China's bone coal resource mainly concentrates on south, and statistics shows that south contains V in the scherbinaite coal
2O
5Total reserves reaches 1.1979 hundred million t, is 7 times of domestic vanadium titano-magnetite, than external other national V
2O
5More than the geology total reserves.Bone coal is a kind of sapanthracite inferior, is by after the death of lower bacteria algae bio, piles up to form under reductive condition.Bone coal except that containing flammable matter, compose the metal deposit and non-metallic element have 60 surplus kind.The higher about kind more than 20 that has industrial utilization to be worth of grade as vanadium, molybdenum, silver, gallium, caesium etc., in addition, is gone back harmful elements such as association chromium, arsenic, mercury, copper, lead, zinc, cadmium, so bone coal is a kind of low-grade polymetallic ore.The grade of vanadium in the bone coal, differing greatly in various places, is generally 0.13%～1.2%, less than 60% (environmental science and technology, 2006,29 (12): 16-17) that account for of cut-off grade 0.5%.Yet there are no at present the report that utilizes the biological metallurgy technology to leach vanadium in the bone coal both at home and abroad.China is the abundantest country of bone coal navajoite reserves in the world.Carry out relevant novel process research and have great strategic importance.
Aspect the microorganism leaching vanadium, " containing useless admittedly vanadium extraction technology of vanadium and prospect (metal mine, 2003,10,61-64) ", " V in the microbiological treatment vanadium-containing water
5+, Cr
6+Experimental study, (Sichuan metallurgy, 2006,28 (5) 30-32) ", " climb the steel test and obtain initial success with vanadium, chromium ion in the microbiological treatment vanadium-containing water, (Shanghai metal, the 29th volume, 62 pages) " wait and microorganism to be soaked vanadium has been done prediction and desk study is studied.
PSM11-the 5th, a strain efficiency natural phosphorus decomposing that obtains and the Penicillium notatum that vanadium, nickel, cobalt etc. in the metallic ore is had the leaching effect are separated in this laboratory.Have wide development, application prospect.
Summary of the invention
The objective of the invention is to be to provide a kind of Penicillium notatum that insolubility phosphorus and metallic ore is had decomposition.This bacterial strain has leaching effect efficiently to metals such as the phosphorus in the Rock Phosphate (72Min BPL) and the nickel in the metallic ore, cobalt, vanadium.
Another object of the present invention is to be to provide the application of a kind of Penicillium notatum in soaking the ore deposit, with the related fungi of this culture medium culturing, can be quickly and efficiently metals such as phosphorus and nickel, cobalt, vanadium be leached in ore, used deposit impregnating technology is simple, cost is low, free from environmental pollution.
To achieve these goals, the present invention adopts following technical measures:
Penicillium (Penicillium) fungi PSM11-5 is that a strain is separated from Chinese navajoite sample by this laboratory and is indication compound with insoluble tricalcium phosphate and sodium metavanadate, cobaltous hydroxide, basic nickel carbonate, decomposes the ability of tricalcium phosphate and sodium metavanadate, cobaltous hydroxide, basic nickel carbonate and the fungal bacterial strain that filters out through test.According to the pedotheque sequence number that separates this bacterial strain, and add that phosphorus dissolves English several PSM of writing a Chinese character in simplified form of microorganism (Phosphatesolubilizing microorganism), with this bacterial strain called after PSM11-5 (Penicillium notatum PSM11-5, Penicillium sp.PSM11-5 CCTCCM208207).Basic know-why is: by playing local method, in PDA substratum (potato substratum), separate the microorganism in the pedotheque, containing insoluble indication compound tricalcium phosphate and sodium metavanadate, cobaltous hydroxide, isolating microorganism in the agar screening culture medium of basic nickel carbonate, formation situation according to indicator dissolving circle, select and to form the obviously bacterial strain of dissolving circle, with the inoculation picked out in the liquid nutrient medium that contains above-mentioned insoluble indication compound, cultivate after 7 days, measure titanium pigment and metal ion nickel in the nutrient solution, cobalt, content of vanadium and pH value.Screen acid producing ability the strongest, to phosphorus, nickel, cobalt, the best fungal bacterial strain PSM11-5 of vanadium leaching effect.
A kind of Penicillium notatum that insolubility phosphorus and metallic ore are had Decomposition, phosphorus, nickel, cobalt, vanadium etc. leach separation, the screening of bacterial strain, and its step is as follows:
A, the sample that will gather from the Guangxi China navajoite are fine ground, and about 5 gram samples place sterile petri dish, pour 33-37 ℃ solid inorganic salt basic medium into, cultivate 5-9 days down at 28-32 ℃, separate single bacterium colony.Consisting of of this solid inorganic salt basic medium (according to document agricultural system science and synthetic study, 2003,19 (4): 260-263): glucose 10 grams, sodium-chlor 0.3 gram, Repone K 0.3 gram, magnesium sulfate heptahydrate 0.3 gram, iron vitriol 0.03 gram, four water manganous sulfates, 0.03 gram ammonium sulfate, 0.5 gram, 15g agar, add distilled water to 1000 milliliters of volumes, regulate pH7.0, add 0.5-1.0% tricalcium phosphate (weight/volume) again;
B, isolating single bacterium colony is switched in four kinds of solid inorganic salt basic medium flat boards of sodium metavanadate, 1-2.0% (weight/volume) cobaltous hydroxide, 1-1.5% (weight/volume) basic nickel carbonate of the tricalcium phosphate that contains 0.5-1.0% (weight/volume) and 1-1.5% (weight/volume), cultivated 5-9 days down at 28-32 ℃.In flat board, form the situation that dissolving is enclosed according to indication compound, select and to form the obviously bacterial strain of dissolving circle;
C, concussion (150 rev/mins) was cultivated 6-8 days in containing the liquid inorganic salt basic medium of above-mentioned insoluble indication compound again, measured titanium pigment or metal ion nickel, cobalt, content of vanadium and pH value (generally 2.2-3.5) in the nutrient solution.Screen acid producing ability the strongest, to phosphorus, nickel, cobalt, the best fungal bacterial strain of vanadium leaching effect, according to the pedotheque sequence number that separates this bacterial strain, and add that phosphorus dissolves English several PSM of writing a Chinese character in simplified form of microorganism (Phosphate solubilizing microorganism), with this bacterial strain called after PSM11-5.(Penicillium notatum PSM11-5, Penicillium sp.PSM11-5CCTCCM208207).
The application primary process of a kind of Penicillium notatum PSM11-5 in soaking the ore deposit:
Utilize to optimize substratum OPSGM and carry out PSM11-5 and soak the ore deposit mode and test, ground phosphate rock content is 20 grams per liters in the substratum.Test design scheme and the results are shown in Table 2 is measured the content and the pH value of titanium pigment in the nutrient solution of each leaching method.Be respectively to cultivate after 3 days and add the ore deposit shaking culture, cultivate and add the static cultivation in ore deposit after 3 days, cultivate and add the ore deposit shaking culture after 7 days and add the static cultivation in ore deposit after 7 days with cultivation.The time of measuring solubility content in the substratum is divided into: before adding the ore deposit, add the ore deposit after, add behind the ore deposit 24 hours, add behind the ore deposit 72 hours, the last adding behind the ore deposit 120 hours and adding the ore deposit 168 hours.Described substratum (OPSGM) [prescription: glucose 50 grams per liters, ammonium sulfate 0.5 grams per liter, sodium-chlor 0.3 grams per liter, Repone K 0.3 grams per liter, manganous sulfate 0.03 grams per liter and yeast extract 1.0 grams per liters] in, the ground phosphate rock or 20 grams per liter bone coal navajoites or the 20 grams per liter serpentinites that add 20 grams per liters were cultivated 7-15 days in 28-32 ℃ of vibration (150 rev/mins) behind the inoculation PSM11-5.Its step is (soaking the ore deposit method):
A1: in the OPSGM substratum, add the ground phosphate rock of 20 grams per liters, cultivated seven days in 30 ℃ of vibrations (150 rev/mins) behind the inoculation PSM11-5, left standstill subsequently two days, measure the content and the pH value of titanium pigment in the nutrient solution.
A2: add the ground phosphate rock of 20 grams per liters in the OPSGM substratum, 30 ℃ leave standstill cultivation seven days behind the inoculation PSM11-5, leave standstill subsequently two days again, measure the content and the pH value of titanium pigment in the nutrient solution.
B1:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated three days, and the ground phosphate rock that adds 20 grams per liters subsequently is shaking culture five days again, measured the content and the pH value of titanium pigment in the nutrient solution.
B2:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated three days, and the ground phosphate rock that adds 20 grams per liters subsequently leaves standstill to be cultivated five days, measured the content and the pH value of titanium pigment in the nutrient solution.
C1:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated seven days, added the ground phosphate rock shaking culture five days of 20 grams per liters subsequently, measured the content and the pH value of titanium pigment in the nutrient solution.
C2:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated seven days, and the ground phosphate rock that adds 20 grams per liters subsequently leaves standstill to be cultivated five days, measured the content and the pH value of titanium pigment in the nutrient solution.
By on draw PSM11-5 soak the ore deposit more suitable soak the ore deposit method: in the OPSGM substratum, add the ground phosphate rock of 20 grams per liters, 30 ℃ of vibrations (150 rev/mins) cultivation seven days, left standstill subsequently two days behind the inoculation PSM11-5.
Invention advantage and effect
Penicillium purpurogenum Penicillium purpurogenum PSM11-5 has very strong leaching effect to metals such as the nickel in vanadium, serpentinite and the laterite in the phosphorus in the Rock Phosphate (72Min BPL), the alum ore, cobalts; thereby can utilize this bacterial strain to carry out biology and soak phosphorus and biological metallurgy; adopt, metals such as phosphorus and vanadium, nickel, cobalt leached the difficult choosing, difficult smelting ore deposit from lean ore, abandoned mine, unbalanced-ore and difficulty, thus reach make full use of Mineral resources, reduce metallurgical cost and energy consumption, purpose such as preserve the ecological environment.Utilize PSM11-5 from low grade rock phosphate, to leach phosphorus, and make during bio-feritlizer is manured into soil, not only can make and contain the higher titanium pigment that can be utilized by farm crop in the soil, this bacterial strain can also leach the insolubility phosphorus that deposits in the past in the soil simultaneously, thereby reduced the usage quantity of phosphate fertilizer, reduced because of producing gas contamination and the water pollutions that phosphate fertilizer brought because of using phosphate fertilizer to bring in a large number, reached Sustainable development, good economy and ecological benefits are arranged, so this bacterial strain has a good application prospect.
(1) PSM11-5 is to the leaching effect of vanadium in the navajoite
(2) PSM11-5 is to the leaching effect of phosphorus in the phosphorus ore
Embodiment
Embodiment 1:
A kind of Penicillium notatum that insolubility phosphorus and metallic ore are had Decomposition, phosphorus, nickel, cobalt, vanadium etc. leach separation, the screening of bacterial strain.Concrete steps are as follows:
A, the sample that will gather from the Guangxi China navajoite are fine ground, and about 5 gram samples place sterile petri dish, pour 33 or 34 or 35 or 36 or 37 ℃ solid inorganic salt basic medium into, cultivate 7 days down at 30 ℃, separate single bacterium colony.Consisting of of this solid inorganic salt basic medium (according to document agricultural system science and synthetic study, 2003,19 (4): 260-263): glucose 10 grams, sodium-chlor 0.3 gram, Repone K 0.3 gram, magnesium sulfate heptahydrate 0.3 gram, iron vitriol 0.03 gram, four water manganous sulfates, 0.03 gram ammonium sulfate, 0.5 gram, 15g agar, add distilled water to 1000 milliliters of volumes, regulate pH7.0, add 0.5% tricalcium phosphate (weight/volume) again;
B, isolating single bacterium colony is switched in four kinds of solid inorganic salt basic medium flat boards of sodium metavanadate, 1% (weight/volume) cobaltous hydroxide, 1% (weight/volume) basic nickel carbonate of the tricalcium phosphate and 1% (weight/volume) that contains 0.5% (weight/volume), cultivated 7 days down at 30 ℃.In flat board, form the situation that dissolving is enclosed according to indication compound, select and to form the obviously bacterial strain of dissolving circle;
C, concussion (150 rev/mins) was cultivated 7 days in containing the liquid inorganic salt basic medium of above-mentioned insoluble indication compound again, measure titanium pigment or metal ion nickel, cobalt, content of vanadium and pH (general 2.2-3.5) value in the nutrient solution, screen acid producing ability the strongest, to phosphorus, nickel, cobalt, the best fungal bacterial strain of vanadium leaching effect, according to the pedotheque sequence number that separates this bacterial strain, and add that phosphorus dissolves English several PSM of writing a Chinese character in simplified form of microorganism (Phosphate solubilizing microorganism), with this bacterial strain called after PSM11-5.(Penicillium notatum PSM11-5, Penicillium sp.PSM11-5CCTCCM208207).
A kind of Penicillium notatum PSM11-5, the application process of Penicillium sp.PSM11-5 CCTCCM208207 is: phosphorus, nickel, cobalt, vanadium etc. leach the evaluation of fungal bacterial strain PSM11-5;
The cultural characteristic of a.PSM11-5 and morphological characteristic:
Penicillium purpurogenum or purple stain mould Penicillium purpurogenum Stoll) PSM11-5 easily cultivates, can grow fast in multiple substratum.Not strict to temperature requirement, at 25-45 ℃ of equal energy normal growths, but at 28-35 ℃ of well-growns.At basic medium (glucose 4.0%, peptone 1.0%, solid medium adds the agar of 1.8%-2%) on the flat board during growth, bacterium colony is velvet-like, produces spore face look and presents faint yellow from the edge to the center successively, dark green, greyish-green, after become blackish green, the back side presents orange, orange red, red-purple.The conidium bar is short and smooth, branch brokenly, and little bar lanceolar forms typical penicillus on the top.Conidium is oval to subsphaeroidal.
The molecular biology identification of b.PSM11-5:
Genomic dna with PSM11-5 is a template, utilize the universal primer (NS1:5-GTATCATATGCTTGTCTC-3 of fungi 18S rDNA, NS8:5-TCCGCAGGTTCACCTACGGA-3) (Agricultural University of the Inner Mongol's journal, 2003,24 (4): 41-44), adopt round pcr to carry out the 18S rDNA amplification of PSM11-5 by follow procedure: 94 ℃ of sex change 5 minutes, then 94 ℃ 30 seconds, 50 ℃ 1 minute 30 seconds, 72 ℃ 2 minutes, 35 circulations; Last 72 ℃ were extended 7 minutes.The PCR product reclaims test kit with DNA glue and reclaims through 1.2% agarose gel electrophoresis, and the PCR product of recovery is by Invitrogen BiotechnologyCo., and Ltd. checks order.The 18S rDNA partial sequence of the PSM11-5 that obtains is the nucleotide sequence shown in the SEQ ID NO.1.
This SEQ ID NO.1 sequence shows that through NCBI BLAST biological software compare of analysis the 18S rDNA similarity of the 18SrDNA of PSM11-5 and Penicillium notatum Penicillium purpurogenum is the highest, reaches 99%.
PSM11-5 detects the resistance of several heavy metal species:
PSM11-5 is to Cd
2+, Co
2+The resistance that higher degree is arranged is to Cr
6+, Hg
2+Moderate resistance is arranged, can be at the CdSO that contains 1300mg/l (weight/volume) respectively
4, 800mg/l (weight/volume) CoCl
280mg/l K
2Cr
2O
7With 80mg/l HgCl
2Substratum in grow; But to Ag
+Sensitivity is at the AgNO that contains 46mg/l (weight/volume)
3Can not grow in the substratum.
The organic acid CHARACTERISTICS IDENTIFICATION that PSM11-5 produces:
Detect the organic acid that PSM11-5 is produced with capillary electrophoresis method in soaking the ore deposit process.
Capillary electrophoresis system: PrinCE 560 (Prince Technologies)
Kapillary: glass capillary, useful length 60cm, internal diameter 50 μ m
Damping fluid: 15 or 20mM PDC (2,6 pyridine dicarboxylic acids 2,6-Pyri dinedicarnoxylicacid) ionogen as a setting, 5mM CTAH (cetyltrimethylammonium hydroxide Cetyltrimethylammonium hydroxide methanol solution) is as electroosmotic flow changed course agent, 1M NaOH adjust pH 5.60, ultrasonic degas is standby behind the damping fluid 0.22 μ m membrane filtration.
Detect: the 230nm ultraviolet detection.
UV-detector: Lambda 1010 (Bischoff) wavelength region: 190-800mm (becoming accurate deuterium lamp) can establish reference wavelength
Described analysis software is: DAX 8.0 Data acquisition ﹠amp; Analysis
Sample preparation: get behind the leach liquor 0.22 μ m membrane filtration after 100 times of the dilutions standby.
Electrophoretic voltage :-20kv
Sample introduction: pressure sample introduction 75Mbar, 6s
Electrophoretic procedures: use 0.1M NaOH flushing 10 minutes first, damping fluid flushing 10 minutes, the damping fluid flushing is 5 minutes between per two sample electrophoresis.
Detected result: the capillary electrophoresis result shows, leaches in the process of phosphorus in the phosphorus ore at PSM11-5, and the organic acid of generation is mainly gluconic acid.
PSM11-5 Optimum of culture medium and soak the ore deposit method
With the upgrowth situation of PSM11-5, to the turnover effect of phosphorus and metal and leaching velocity etc. is leading indicator, by kind screening, content optimization to carbon source in the substratum and nitrogenous source, acquisition is suitable for PSM11-5 growth, leaching efficiency height, fireballing excellent culture medium (OPSGM) prescription: glucose 50 grams per liters, ammonium sulfate 0.5 grams per liter, sodium-chlor 0.3 grams per liter, Repone K 0.3 grams per liter, manganous sulfate 0.03 grams per liter and yeast extract 1.0 grams per liters, tricalcium phosphate 5 grams per liters or ground phosphate rock 20 grams per liters.
(1) Penicillium notatum (fungi) bacterial strain PSM11-5 leaching effect to tricalcium phosphate and phosphorus ore in OPSGM compares
PSM11-5 is cultivated in 30 ℃ of vibrations (150 rev/mins) in the optimization substratum OPSGM that contains 5 grams per liter tricalcium phosphates and 20 grams per liter ground phosphate rock respectively, measure titanium pigment content 59.2-966.4 and pH3.1-5.8 value and titanium pigment content 52.5-300.1 and pH3.2-6.3 in the nutrient solution, see Table 1
Table 1 PSM11-5 changes pH value in the leaching effect of phosphorus in tricalcium phosphate and the Rock Phosphate (72Min BPL) and the nutrient solution
(2) PSM11-5 soaks the ore deposit mode
Utilize to optimize substratum OPSGM and carry out PSM11-5 and soak the ore deposit mode and test, ground phosphate rock content is 20 grams per liters in the substratum.Test design scheme and the results are shown in Table 2 is measured the content and the pH value of titanium pigment in the nutrient solution of each leaching method.Be respectively to cultivate after 3 days and add the ore deposit shaking culture, cultivate and add the static cultivation in ore deposit after 3 days, cultivate and add the ore deposit shaking culture after 7 days and add the static cultivation in ore deposit after 7 days with cultivation.The time of measuring solubility content in the substratum is divided into: before adding the ore deposit, add the ore deposit after, add behind the ore deposit 24 hours, add behind the ore deposit 72 hours, the last adding behind the ore deposit 120 hours and adding the ore deposit 168 hours.
Soak the ore deposit method:
A1: in the OPSGM substratum, add the ground phosphate rock of 20 grams per liters, cultivated seven days in 30 ℃ of vibrations (150 rev/mins) behind the inoculation PSM11-5, left standstill subsequently two days, measure the content and the pH value of titanium pigment in the nutrient solution.
A2: add the ground phosphate rock of 20 grams per liters in the OPSGM substratum, 30 ℃ leave standstill cultivation seven days behind the inoculation PSM11-5, leave standstill subsequently two days again, measure the content and the pH value of titanium pigment in the nutrient solution.
B1:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated three days, and the ground phosphate rock that adds 20 grams per liters subsequently is shaking culture five days again, measured the content and the pH value of titanium pigment in the nutrient solution.
B2:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated three days, and the ground phosphate rock that adds 20 grams per liters subsequently leaves standstill to be cultivated five days, measured the content and the pH value of titanium pigment in the nutrient solution.
C1:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated seven days, added the ground phosphate rock shaking culture five days of 20 grams per liters subsequently, measured the content and the pH value of titanium pigment in the nutrient solution.
C2:PSM11-5 30 ℃ of vibrations (150 rev/mins) in not containing the OPSGM substratum of ground phosphate rock were cultivated seven days, and the ground phosphate rock that adds 20 grams per liters subsequently leaves standstill to be cultivated five days, measured the content and the pH value of titanium pigment in the nutrient solution.
Table 2, fungi PSM11-5 soak ore deposit method and effect relatively in the OPSGM substratum
By on draw PSM11-5 soak the ore deposit more suitable soak the ore deposit method: in the OPSGM substratum, add the ground phosphate rock of 20 grams per liters, 30 ℃ of vibrations (150 rev/mins) cultivation seven days, left standstill subsequently two days behind the inoculation PSM11-5.
SEQUENCE?LISTING
＜110〉Wuhan Virology Institute,Chinan academy of Sciences
＜120〉a kind of Penicillium notatum and preparation method and application
＜130〉a kind of Penicillium notatum and preparation method and application
<160>1
<170>PatentIn?version?3.1
<210>1
<211>1669
<212>DNA
＜213〉Penicillium notatum
<400>1
Claims (2)
1, a kind of Penicillium notatum is characterized in that: Penicillium sp.PSM11-5 CCTCCM208207.
2, the application of the described a kind of Penicillium notatum of claim 1 in soaking the ore deposit.
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