CN110951626B - Penicillium oxalicum capable of decomposing coal gangue - Google Patents
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Abstract
The invention discloses a strain Penicillium oxalicum (Penicillium oxalicum) for decomposing coal gangue, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No. 7699. The invention also discloses a solid substrate formula of the high-yield spore of the strain. The bacterial strain has the best dissolving effect on insoluble phosphate at the culture temperature of 28 ℃, 1-5% of the bacterial liquid addition amount in a liquid culture system has different decomposition effects on coal gangue, and plants are planted in the coal gangue matrix added with the penicillium oxalicum bacterial liquid, so that the seed germination rate, the plant biomass, the available nutrients and the microbial biomass can be improved. Therefore, the invention can provide excellent strain resources for resource utilization of coal gangue.
Description
Technical Field
The invention belongs to the technical field of environmental biology, and particularly relates to a penicillium oxalicum capable of tolerating heavy metals, efficiently dissolving phosphorus and decomposing coal gangue.
Technical Field
The coal gangue is a solid waste in the coal mining process, the comprehensive emission accounts for 10% -15% of the raw coal output, and the coal gangue is one of the industrial and mining solid wastes with the largest emission in China. The large stacking of the coal gangue not only occupies a large amount of land resources, but also causes the release of toxic heavy metal elements such as lead, cadmium, chromium and the like in the coal gangue to the surrounding environment and causes environmental pollution due to the continuously generated acidic wastewater of the coal gangue under the action of natural oxidation and wind-blowing and rain-showering. At present, the main treatment mode of the coal gangue is landfill, and the resource utilization of the coal gangue mainly comprises the aspects of paving, producing building materials, burning and generating electricity, manufacturing artware and the like. In addition, coal gangue power generation, industrial refining of alumina and silicon products, rock wool, sulfur and the like, brick making in the building industry, cement and the like are also available. The common features of the above solutions: large project investment, high production cost, secondary waste generation, no advantages of products, strong substitution of similar products and the like.
The determination result of the coal gangue shows that the coal gangue contains a certain amount of organic matters, phosphorus, potassium and a plurality of trace elements, and the crushed coal gangue or weathered matters have certain physical characteristics of soil, such as coarse particles, large pores, high permeability coefficient, low field water holding capacity, low wilting coefficient and accumulated evaporation capacity, certain water retention performance, high utilization rate of available water and low available nutrients. After the coal gangue is treated by physical, chemical or biological treatment, the physical properties of the coal gangue are improved or the effective nutrient content and the utilization rate of the coal gangue are improved, so that the coal gangue becomes a substrate or soil for plant growth. Chinese patent (publication No. CN109438116A) discloses a method for preparing a soil conditioner by modifying and activating coal gangue through high-temperature roasting (600-900 ℃), wherein the coal gangue is roasted under high-temperature conditions when the modified coal gangue is prepared, so that the energy consumption and the cost are greatly increased.
The microorganisms generally exist in nature, have the characteristics of small volume, quick propagation, strong adaptability, wide food spectrum and the like, and play an irreplaceable role in the resource utilization of wastes and the environmental pollution treatment. The fungus group is widely distributed in nature and has extremely strong adaptability to the environment, for example, Chinese patent (publication number: CN103923839A) discloses a heavy metal tolerant penicillium which is separated from a coal mine area, has tolerance to various heavy metals and has important significance for modifying the heavy metal pollution of coal gangue. The research shows that the trichoderma has the decomposition effect on coal gangue, and hypha of the trichoderma can extend into minerals to obtain nutrition, so that the crushing and decomposition of the minerals are promoted. Therefore, aiming at the characteristics of the coal gangue waste, the microorganisms capable of tolerating heavy metals in the coal gangue and dissociating the insoluble phosphorus of the coal gangue to decompose the coal gangue are screened, and the method has important significance for resource utilization of the coal gangue.
The strain related to the patent is a high-efficiency penicillium oxalicum separated and screened from lead-zinc tailings in Huayuan county of Hunan province, and the strain is used for Pb2+、Zn2+、Cr2+、Mn2+The strain has applied for Chinese patent (publication number: CN103614302A), and further research shows that the strain has the function of decomposing coal gangue. At present, researches on penicillium oxalicum which is tolerant to heavy metals and has the effects of efficiently dissolving phosphorus and decomposing coal gangue are not reported at home, and the development of the bacterial strain has important significance for resource utilization of the coal gangue, improvement of physicochemical properties of the coal gangue and promotion of plant growth.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the penicillium oxalicum capable of decomposing the coal gangue is provided, and the penicillium oxalicum can dissolve insoluble phosphorus in the coal gangue and decompose the coal gangue, thereby promoting plant growth and improving substrate nutrient and microbial biomass.
The technical scheme provided by the invention is as follows: a strain of Penicillium oxalicum (Penicillium oxalicum) TJ2 with the preservation number of CGMCC No.7699 for decomposing coal gangue is preserved in the China general microbiological culture Collection center.
The Penicillium oxalicum (Penicillium oxalicum) TJ2 is preserved in China general microbiological culture Collection center (CGMCC) in 2013, 6 and 14 months (the preservation address is No. 3 Hospital No. 1 of Xilu, Beijing, Chaoyang, Beicheng, the postal code is 100101), the preservation number is CGMCC No.7699, and the Penicillium oxalicum (Penicillium oxalicum) survives after detection.
The invention relates to the culture of the strain, and the culture medium formula is as follows: 50 parts of soybean cake powder and 50 parts of corn powder50 parts of bran and inorganic salt mixed solution, the weight of which is 1 percent of the total weight of the solid matrix, the amount of distilled water which is 25 percent of the total weight of the soybean cake powder and the bran, the natural pH value and the inoculation are 108The inoculation amount of bacterial liquid of/ml is 5 percent, the culture medium and the bacterial liquid are uniformly stirred, the mixture is placed in an incubator at the temperature of 28 ℃ for culture for 5 days, and then the mixture is cultured according to the ratio of culture to sterile water 1: 10, then placing the mixture in a shaking table at 150rpm for 1h, taking out the mixture, standing the mixture for 10min, and counting the content of the penicillium oxalicum spores according to a blood counting plate method for subsequent experiments.
The bacterial strain of the invention dissolves insoluble calcium phosphate at different culture temperatures, the spore suspension of the penicillium oxalicum is taken, and the volume ratio of the spore suspension to a liquid phosphate dissolving culture medium (10 g/L of cane sugar, 10g/L of calcium phosphate, 0.3g/L of sodium chloride, 0.3g/L of potassium chloride, 0.3g/L of magnesium sulfate, 0.03g/L of ferrous sulfate, 0.03g/L of manganese sulfate, 0.5g/L of ammonium sulfate, 0.5g/L of yeast extract, pH7.2) is 1: 20 inoculation, and sampling to determine the content of soluble phosphorus after 5 days of culture.
The bacterial strain of the invention has the decomposition effect on coal gangue, 50g of coal gangue crushed into 18 meshes is added into three bottles of 250ml, distilled water with the same amount is added, a certain amount of penicillium oxalicum spore suspension is added into the system, and four addition amounts are set: only adding distilled water (CK), low-dose penicillium oxalicum (1%), medium-dose penicillium oxalicum (2.5%) and high-dose penicillium oxalicum (5%), culturing in a shaker at the rpm of 180 r and the culture temperature of 28 ℃ for 15 days, and determining the coal gangue decomposition effect of the strain of the invention by measuring the weight reduction of coal gangue and the content of efficient phosphorus in a culture solution.
The bacterial strain disclosed by the invention has the effect of coal gangue on plant growth, crushed coal gangue powder passing through an 8-mesh sieve is taken, 1%, 2.5% and 5% of the weight of the crushed coal gangue powder is added with penicillium oxalicum liquid, the crushed coal gangue powder and the penicillium oxalicum liquid are uniformly mixed by taking the treatment without adding the bacterial liquid as a control, the mixture is put into a flowerpot with the volume of 1L, the same number of ryegrass seeds are planted, the bacterial liquid is poured at different times every week according to different treatment, the germination rate of the seeds is observed, and the biomass of the ryegrass, the nutrient content of the coal gangue and the microbial biomass.
The invention has the following beneficial effects:
the Penicillium oxalicum (Penicillium oxalicum) TJ2 is from a heavy metal polluted mining area, and the highest-concentration spore suspension is obtained by solid state fermentation of soybean cake powder and bran. The bacterium has the function of converting insoluble phosphorus into soluble phosphorus, the soluble content is 2053.2mg/ml in the treatment of adding the penicillium oxalicum bacterium liquid, and the soluble phosphorus content in a control system without the bacterium is only 73.4mg/ml, which shows that the strain has high-efficiency phosphate solubilizing effect.
The bacterial strain is added into a liquid culture system containing gangue powder in different dosages, and compared with the treatment without the bacterial strain, the bacterial strain can decompose gangue to different degrees and increase the content of available phosphorus in the system. The treatment of adding the penicillium oxalicum bacterial liquid into the coal gangue to plant the ryegrass can improve the germination rate of seeds, and the nutrient content, the ryegrass biomass and the microorganism biomass in the coal gangue are obviously higher than those of a control, which shows that the bacterial strain can decompose the coal gangue, release nutrients and improve the microorganism content, thereby being beneficial to the growth of plants. The invention provides theoretical and technical support for resource utilization of coal gangue.
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FIG. 1 Effect of different solid substrates on the sporulation of Penicillium oxalicum
FIG. 2 Effect of adding Penicillium oxalicum to the culture solution of inorganic salts on spore production
Detailed Description
The present invention will be better understood by those skilled in the art from the following examples. The examples are described for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1: solid state fermentation for decomposing penicillium oxalicum of coal gangue
The mold is generally easier to produce a large amount of spores in a solid culture medium, and the large amount of spores of the penicillium oxalicum provides a large amount of strains for the subsequent application of coal gangue decomposition. The study shows that the difference of the solid medium components has a great influence on the spore yield, so the present example studies the solid fermentation medium components of the strain of the present invention. The solid culture medium comprises bran, soybean cake powder, corn flour, bran and soybean cake powder 1: 2. branMixing with corn flour 1: 2. soybean cake powder and corn flour 1: 1. and bran, soybean cake flour, corn flour 1: 1: 1, marked as treatment 1-treatment 7, placed in 500ml triangular flasks, each filled with 20g of the total weight of the solid medium, 25% of distilled water, 2.5% of the weight of the system, at a concentration of 1X 108Standing and culturing spore suspension of each spore/ml in an incubator at 28 ℃, stirring the solid culture medium by using a sterile glass rod every two days for 5 days, and then mixing the culture with sterile water 1: 10, adding the mixture, placing the mixture into a rotary table rotating at the rpm of 150 to oscillate for 1h, taking out the mixture, standing the mixture for 10min, and counting the content of the penicillium oxalicum spores according to a blood counting plate method. The solid culture medium with the largest spore yield is used as a formula raw material, and 1%, 3% and 5% of inorganic salt culture solution (formula: K in 1L of distilled water) is added2HPO4 0.2g,KH2PO4 0.8g,MgSO4 0.2g,CaSO4·0.5H2O 0.1g,Na2MoO4·5H2O 0.0033g,FeSO4·7H2O0.005 g, pH7.2), and the culture solution without addition of inorganic salts were used as controls, and the inoculum size, culture conditions, operation during culture, and the like were the same as those in the previous experiment, and the influence of different amounts of inorganic salts on the spore yield of the solid medium was measured. The results show that the wheat bran, soybean cake powder and corn flour are 1: 1: the solid culture medium with the formula 1 has the largest spore yield which reaches 5.76 multiplied by 109The spore yield can be improved by adding inorganic salt culture medium (see figure 1), wherein the spore yield is highest when 3% of inorganic salt is added, and reaches 9.52 × 109Spores per gram of matrix (see FIG. 2).
FIG. 1 Effect of different solid substrates on the sporulation of Penicillium oxalicum
FIG. 2 Effect of adding Penicillium oxalicum to the culture solution of inorganic salts on spore production
Example 2: influence of temperature on the dissolution of insoluble phosphates in penicillium oxalicum
Selecting 5 temperature gradients, adding calcium phosphate as insoluble phosphate, and preparing the following culture medium, wherein the formula of the culture medium is as follows: glucose 10.0g, (NH)4)2SO4 0.5g，MgSO4·7H2O 0.3g，NaCl 0.3g，KCl 0.3g，FeSO40.03g，MnSO4·H20.03g of O, 0.5g of yeast powder and Ca3(PO4)210g of distilled water l000ml, pH 7.0. 50ml of solution is filled in each bottle, and 5 percent of penicillium oxalicum spore suspension (with the concentration of 1 multiplied by 10) is added7The culture temperature is set at 20 ℃, 24 ℃, 28 ℃, 32 ℃ and 36 ℃, the culture time is 5 days, the fermentation liquor is centrifuged for 10min at 4 ℃ and 10000r/min, and the content of soluble phosphorus in the fermentation liquor is measured by a molybdenum-antimony colorimetric method, and the result is shown in table 1. As can be seen from Table 1, the phosphorus dissolving effect of the penicillium oxalicum is different with different culture temperatures, wherein the phosphorus dissolving effect is the best at the culture temperature of 28 ℃, and the content of soluble phosphorus in the system reaches 2284.7 mg/L.
TABLE 1 Effect of different culture temperatures on the dissolution of insoluble phosphorus by Penicillium oxalicum
| Temperature (. degree.C.) | 20 | 24 | 28 | 32 | 36 |
| Soluble phosphorus content (mg-L) | 1736.5 | 1902.4 | 2284.7 | 2105.1 | 1851.8 |
Example 3: decomposition of coal gangue by penicillium oxalicum
Pulverizing coal gangue, sieving with 18 mesh sieve, weighing 50g undersize, placing in 250ml triangular flask, adding equal amount of distilled water, and suspending penicillium oxalicum spore with concentration of 1.28 × 107And/ml) is added into the system according to the proportion of 1 percent, 2.5 percent and 5 percent, the treatment without adding bacterial suspension is taken as a reference, the shaking culture is carried out for 15 days in a shaking table with the rpm of 180 revolutions and the culture temperature of 28 ℃, collected liquid is filtered by filter paper and then is centrifuged at 8000g, the supernatant is used for measuring soluble phosphorus, undecomposed coal gangue powder is collected and weighed after being naturally air-dried, and the decomposition rate of the bacterial strain to the coal gangue is calculated by measuring the weight reduction of the coal gangue. As can be seen from Table 2, the added penicillium oxalicum liquid has different degrees of promotion effects on coal gangue decomposition, and compared with a control, the decomposition rate of coal gangue and the content of soluble phosphorus in each treatment are obviously higher than those in the control, wherein the addition amount of the added penicillium oxalicum liquid is 2.5%, and the decomposition rate of the treated coal gangue and the content of the soluble phosphorus are the highest, and are respectively 18.47% and 89.89 mg/L.
TABLE 2 decomposition of coal gangue by Penicillium oxalicum
Example 4: plant growth promoting effect of adding penicillium oxalicum in coal gangue
Taking the pulverized coal gangue powder which is sieved by an 8-mesh sieve, adding penicillium oxalicum liquid according to 1%, 2.5% and 5% of the weight, taking the treatment without adding the bacterial liquid as a control, uniformly mixing the pulverized coal gangue powder with the pulverized coal gangue powder, putting the mixture into a flowerpot with the volume of 1L, planting 100 ryegrass seeds, pouring the bacterial liquid at different treatment timings every week, observing the germination rate of the seeds, and measuring the biomass, nutrient content and microbial biomass of the ryegrass after 50 days of planting. As can be seen from Table 3, the addition of the penicillium oxalicum liquid has different degrees of promotion effects on the germination rate of ryegrass seeds, plant biomass, available nutrients of a matrix and microbial biomass, and compared with a control, the indexes of each treatment are obviously higher than that of the control, wherein the treatment difference between the addition amount of 2.5% and 5% is not obvious, which shows that the addition amount of 2.5% of the penicillium oxalicum liquid can play a significant role.
TABLE 3 influence of Penicillium oxalicum on coal gangue particle size composition
Claims (3)
1. A strain of penicillium oxalicum (Penicillium oxalicum) The application of the penicillium oxalicum stored in the common microorganism center of China Committee for culture Collection of microorganisms and management, with the preservation number of CGMCC number 7699, in promoting the growth of ryegrass; the soil for the growth of the ryegrass contains coal gangue.
2. Use according to claim 1, characterized in that: adding the penicillium oxalicum liquid and coal gangue into soil when the ryegrass seeds are sowed, and pouring the penicillium oxalicum liquid regularly every week.
3. Use according to claim 2, characterized in that: the adding amount of the penicillium oxalicum bacterial liquid is 1-5% of the coal gangue by weight.
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