CN108587947B

CN108587947B - Phosphate solubilizing bacteria, composite microbial inoculum of phosphate solubilizing bacteria and DEHP degrading bacteria and application of phosphate solubilizing bacteria and DEHP degrading bacteria in soil improvement

Info

Publication number: CN108587947B
Application number: CN201810316362.XA
Authority: CN
Inventors: 莫测辉; 冯乃宪; 喻乐意; 李彦文; 蔡全英; 赵海明; 李慧
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-04-10
Filing date: 2018-04-10
Publication date: 2020-05-08
Anticipated expiration: 2038-04-10
Also published as: CN108587947A

Abstract

The invention discloses a phosphorus-dissolving bacterium, a composite microbial inoculum of the phosphorus-dissolving bacterium and a DEHP degrading bacterium, and application of the composite microbial inoculum in soil improvement. The strain is Bacillus megaterium YLLYP 1, which is preserved in Guangdong province microorganism strain preservation center in 2017, 06 and 21 months, and the preservation number is as follows: GDMCC NO: 60204. the invention also provides a complex microbial inoculum, which comprises YLVP 1 and Rhodococcus pyrivorans XB. The compound microbial inoculum has good cooperativity, the phosphorus dissolving capacity and the DEHP degrading capacity of the compound microbial inoculum are obviously improved, the culture method is simple and easy to implement, the propagation is rapid, the adaptability is strong, the YLEP 1 and XB can be used for effectively improving the phosphorus deficiency condition of soil and removing DEHP organic pollutants in the soil, and the compound microbial inoculum has important significance for cultivating and improving the ecological fertility of the soil, ensuring the quality safety of agricultural products and realizing production and repair.

Description

Phosphate solubilizing bacteria, composite microbial inoculum of phosphate solubilizing bacteria and DEHP degrading bacteria and application of phosphate solubilizing bacteria and DEHP degrading bacteria in soil improvement

Technical Field

The invention belongs to the technical field of microbial engineering, and particularly relates to a phosphorus-solubilizing bacterium, a compound microbial inoculum of the phosphorus-solubilizing bacterium and DEHP degrading bacteria, and application of the compound microbial inoculum in soil improvement.

Background

Phosphorus (P) is one of the macronutrient elements essential for plant growth and developmentIt is involved in various physiological metabolic activities and morphologies of plants, and determines the yield and quality of crops. The phosphorus required by plants is mainly sourced from soil. Most farmland soil phosphorus deficiency is caused because phosphorus in the soil usually exists in the form of difficultly soluble salt. More than 70 percent of cultivated land in China is lack of phosphorus. In recent years, with the increasing of crop yield, the application amount of phosphate fertilizer is increasing, and the phosphate fertilizer applied to farmland is easy to be Ca-coated²⁺、Fe³+、Al³⁺When cations are combined to form insoluble phosphate for deposition and fixation on soil, the in-season utilization rate of the phosphate fertilizer by crops is only 3.7-32.4%. The large amount of phosphate fertilizer application not only increases the agricultural production cost, but also causes soil hardening and fertility reduction, and causes a series of environmental problems such as water pollution and soil pollution. Therefore, the phosphorus-dissolving bacteria release phosphorus fixed by soil, the use amount of phosphate fertilizer is reduced, the phosphorus-dissolving bacteria is one of effective ways for improving the utilization rate of phosphorus, the phosphorus-dissolving bacteria is an important measure for dealing with the zero-growth policy of chemical fertilizers in China, and the phosphorus-dissolving bacteria have important significance for the sustainable development of agriculture in China.

On the other hand, the pollution of organic pollutants such as Phthalic Acid Esters (PAEs), Polycyclic Aromatic Hydrocarbons (PAHs) and the like in farmland soil is caused by the large discharge of industrial three wastes and the unreasonable use of agricultural products such as chemical fertilizers, pesticides, agricultural films and the like. Organic pollutants such as PAEs in the polluted soil are absorbed and accumulated by crops, and the quality safety of agricultural products is seriously influenced. Organic pollutants such as PAEs belong to environmental endocrine disruptors, can be accumulated in a human body, has reproductive development toxicity, liver toxicity and 'three-cause' effects (carcinogenesis, teratogenesis and mutagenesis), and causes long-term low-dose exposure and serious harm to human health due to the fact that residents eat agricultural products containing the organic pollutants such as the PAEs in a daily way.

Therefore, the research and development of the composite microbial inoculum with the functions of dissolving phosphorus and degrading PAEs have important significance for the current situations of phosphorus deficiency and PAEs pollution of farmland soil in China.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a phosphate solubilizing bacterium.

The invention also aims to provide a complex microbial inoculum.

The invention also aims to provide application of the phosphate solubilizing bacteria and the composite microbial inoculum.

The phosphorus-dissolving bacteria and the composite microbial inoculum thereof provided by the invention can solve the problem of phosphorus deficiency of soil, reduce the DEHP pollution of the soil and ensure the quality safety of agricultural products.

The purpose of the invention is realized by the following technical scheme:

the invention provides a strain of phosphate solubilizing bacteria, which is named as Bacillus megaterium YLLYP 1 and is obtained by separating and purifying activated sludge of a sewage treatment plant.

The phosphate solubilizing bacterium is Bacillus megaterium YLLYP 1, and the preservation information is as follows: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: day 21, 2017, 06 and 21, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60204.

the Bacillus megaterium (Bacillus megaterium) YLLYP 1 has the culture characteristics and morphological characteristics that: the thallus is gram-positive bacteria; spores are generated in the middle or on the partial ends, and the spores are elliptic; observing under an electron microscope to form a long rod, wherein the average length of the thallus is 1.9-2.2 μm, and the average width is 0.7-0.9 μm (figure 1); culturing on LB plate culture medium at 30 deg.C for 24h to obtain yellowish colony, which is flat and raised, and has neat edge, smooth and opaque surface (FIG. 2); the results of the physiological and biochemical identification experiments of the strain YLYP1 are shown in Table 1.

The invention also provides application of the Bacillus megaterium YLLYP 1 in dissolving insoluble phosphate.

The insoluble phosphate is calcium phosphate, aluminum phosphate or iron phosphate.

Preferably, the sparingly soluble phosphate is calcium phosphate.

The Bacillus megaterium YLLYP 1 dissolves insoluble phosphate in NBRIP culture medium until the content of soluble phosphorus reaches 95.3 +/-3.2 mg/L-405.0 +/-3.7 mg/L.

The invention also provides a complex microbial inoculum, which comprises Bacillus megaterium YLVP 1 and Rhodococcus pyritivorans XB.

Rhodococcus pyridinivorans XB separated and screened in the preliminary study of the subject group can effectively reduce diisooctyl phthalate (DEHP) in soil and reduce the absorption and accumulation of DEHP by corn. The preservation information of the strain is as follows: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 2016, 09/28, accession: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCCNO: 60054.

the Bacillus megaterium YLLYP 1 and Rhodococcus pyridinivorans XB are prepared to have a concentration of 10⁸～10¹⁰cfu/mL of bacterial suspension;

the Bacillus megaterium YLLYP 1 and the Rhodococcus pyridinivorans XB are mixed according to the volume ratio of 2-5: 1-3, mixing; preferably, the volume ratio of 2: 1-3, mixing; more preferably, the ratio by volume of 2: 1, mixing.

The preparation method of the complex microbial inoculum comprises the following steps:

(1) respectively inoculating Bacillus megaterium YLVP 1 and Rhodococcus pyridinivorans XB into LB liquid culture medium, and activating at 28-30 ℃ for 12-18 h;

(2) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10⁸～10¹⁰cfu/mL；

(3) 2 kinds of single bacteria are treated according to the following steps of 2-5: and (3) uniformly mixing the components in a volume ratio of 1-3 to obtain the multifunctional composite microbial inoculum.

The composite microbial inoculum is applied to dissolving phosphorus and/or degrading DEHP.

The content of soluble phosphorus of the compound bacterial agent in an NBRIP culture medium is 482.5-570.3 mg/L.

The compound microbial inoculum improves the degradation capability of DEHP in an MSM culture medium containing DEHP, and can basically and completely degrade the DEHP.

The result shows that the content of soluble phosphorus in the culture solution of Bacillus megaterium YLLYP 1 is 405.0 mg/L-436.2 mg/L, the content of soluble phosphorus in the culture solution of Rhodococcus pyritivorans XB is 15.7 mg/L-20.2 mg/L, and the content of soluble phosphorus in the culture solution of the complex microbial inoculum is 482.5 mg/L-570.3 mg/L. The phosphorus dissolving capacity of the composite microbial inoculum is obviously improved, and the compounding effect of the 1+1>2 microbial inoculum is achieved.

Determination of DEHP degradation rate: the Bacillus megaterium YLLYP 1 has no degradation of DEHP in the MSM culture medium containing DEHP, the degradation rate of Rhodococcus dipyridinivorans XB to DEHP in the MSM culture medium containing DEHP is 80.5-91.9%, and the degradation rate of the complex microbial inoculum to DEHP in the MSM culture medium containing DEHP is 90.5-100%. Therefore, the degradation capability of the compound microbial inoculum on DEHP is improved.

The composite microbial inoculum is applied to improving the soil quality.

Preferably, the compound microbial inoculum is inoculated into the soil and uniformly stirred, so that the content of available phosphorus in the soil can be increased, and DEHP in the soil can be degraded at the same time.

Adding 600g of soil into a 500mL beaker, shading with tinfoil paper, adding 60mL of each of the strains YLYP1, XB and the compound microbial inoculum into the soil, and uniformly mixing. Adjusting the water content of the soil to 30% of field water capacity, culturing at 30 ℃ in a dark place at constant temperature for 10 days, and repeating three times for each treatment by taking soil without any microbial inoculum as a control. And (4) measuring the content of available phosphorus in the soil treated by different methods. The result shows that the effective phosphorus content in the contrast soil is 39.8mg/kg, the effective phosphorus content in the inoculated single bacterium Bacillus megaterium YLVP 1 soil is 59.5-68.1 mg/kg, the effective phosphorus content in the inoculated single bacterium Rhodococcus pyritivivorans XB soil is 41.7-45.2 mg/kg, and the effective phosphorus content in the inoculated compound microbial inoculum soil is 76.2-87.2 mg/kg.

The DEHP concentration was set at 20 mg/kg. Adding 600g of soil into a 500mL beaker, adding DEHP, stirring uniformly, shading with tinfoil paper, aging for one week, adding 60mL of the bacterial strains YLYP1, XB and the composite microbial inoculum into the soil, and stirring uniformly. Adjusting the water content of the soil to 30% of the field water capacity, culturing at constant temperature of 30 ℃ for 15d, measuring the residual quantity of DEHP in the soil, and taking the soil without any microbial inoculum as a blank control. Each of the above processes was set to 3 repetitions. The result shows that the degradation rate of DEHP in the soil in the blank control is 27.1 percent, the degradation rate of DEHP in the soil inoculated with single bacterium Bacillus YLYPP 1 is 32.2-36.5 percent, the degradation rate of DEHP in the soil inoculated with single bacterium Rhodococcus pyritivorns XB is 53.9-65.3 percent, and the degradation rate of DEHP in the soil added with the composite microbial inoculum is 70.3-93.7 percent respectively.

Compared with the prior art, the invention has the following advantages and effects:

the multifunctional composite microbial inoculum disclosed by the invention has good cooperativity, the phosphorus dissolving capacity and the DEHP degrading capacity of the composite microbial inoculum are obviously improved, the culture method of the composite microbial inoculum is simple and easy to implement, the propagation is rapid, the adaptability is strong, and the application potential is good.

Drawings

FIG. 1 shows the morphology of Bacillus megaterium YLYP1 cells.

FIG. 2 shows the colony morphology of Bacillus megaterium YLYP 1.

FIG. 3 is a phylogenetic tree of Bacillus megaterium (Bacillus megaterium) YLLYP 1.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Deposit information of the strain Rhodococcus pyritivorans XB used in the examples: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 2016, 09/28, accession: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60054.

inorganic salt medium (MSM, g.L)^-1)：K₂HPO₄5.8，KH₂PO₄4.5，(NH₄)₂SO₄2.0，MgCl₂0.16，CaCl₂0.02，Na₂MoO₄·2H₂O 0.0024，FeCl₃0.0018，MnCl₂·2H₂O0.0015, pH 7.5. The initial concentration of DEHP added to MSM medium was 400mg L^-1As the sole carbon source.

Example 1

Screening of phosphate solubilizing bacteria:

(1) the separation method of the strain is a plate screening method: collecting activated sludge from a sewage treatment plant, placing the activated sludge in a 50mL sterile centrifuge tube, and placing the sterilized activated sludge in a clean ice box. Weighing 1g of activated sludge, dissolving in a 50mL centrifuge tube containing 40mL of sterile water, shaking uniformly by a 130rpm oscillator, and diluting the sample with 0.9% sterile physiological saline to 10^-1～10^-5And (3) solution.

(2) Culture medium:

monkina (PVK) liquid medium: 5.0g of calcium phosphate, 10.0g of cane sugar, 0.5g of ammonium sulfate, 0.1g of sodium chloride, 0.1g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.03g of manganese sulfate, 0.5g of yeast extract and 1000mL of distilled water, wherein the pH value is 7.0-7.2. 15g of agar was added to the PVK solid medium.

Phosphate growth (NBRIP) medium: 10.0g of glucose, 5.0g of calcium phosphate, 5.0g of magnesium chloride hexahydrate, 0.25g of magnesium sulfate heptahydrate, 0.2g of potassium chloride, 0.1g of ammonium sulfate, 1000mL of distilled water and pH 7.0.

LB liquid medium: 5.0g of yeast powder, 10.0g of peptone, 10.0g of sodium chloride, 1000mL of distilled water and pH 7.0. 15g of agar was added to the LB solid medium.

All cultures were sterilized for 20min at 121 ℃.

(3) Screening phosphate solubilizing bacteria: respectively sucking 0.1mL of diluent with different dilution times, uniformly coating the diluent on a PVK solid culture medium, placing the PVK solid culture medium at 30 ℃ for 5 days of inverted culture, selecting a bacterial colony with good growth vigor and obvious phosphate solubilizing ring, transferring the bacterial colony to the PVK solid culture medium by a scribing method, repeating the steps for 2-3 times, and finally obtaining a phosphate solubilizing bacterium with strong phosphate solubilizing capability, wherein the number of the phosphate solubilizing bacterium is YLYP 1. The culture medium is transferred to an LB culture medium test tube inclined plane for preservation in a refrigerator at 4 ℃ and is used for subsequent experiments.

Example 2

Identifying a phosphorus-solubilizing strain YLLYP 1:

the detection basis and method of morphological characteristics and physiological biochemistry is a bacterial identification program MS (i)/C005-C01. And (3) morphological identification: the strain is a gram-positive bacterium and has spores which are mesogenic or subterminal and elliptical; the cells were elongated under an electron microscope, and had an average length of 1.9 to 2.2 μm and an average width of 0.7 to 0.9. mu.m (FIG. 1). After being cultured on LB solid medium at 30 ℃ for 24h, the colonies are milky yellow, flat and raised, smooth and opaque in surface and neat in edge (FIG. 2).

Physiological and biochemical characteristic identification: the results of the physiological and biochemical experiments are shown in Table 1.

TABLE 1 physiological and biochemical characteristics of Bacillus megaterium YLVP 1

Physiological and biochemical test	Results	Physiological and biochemical test	Results
				Contact enzyme	+	Fermentation of D-glucose	-
Oxidase enzyme	-	Fermentation of trehalose	-
				Anaerobic growth	-	Fermentation of mannose	-
V-P assay	-	Fermentation of maltose	-
				Nitrate reduction	-	Rhamnose fermentation	-
Starch hydrolysis	+	Exercise of sports	+
				Liquefaction of gelatin	+	Growth experiment with 2% NaCl	+
Phenylalanine dehydroxylase	-	Growth experiment with 5% NaCl	+
				Citrate utilization	-	7% NaCl growth experiment	-
Lecithinase	-	Growth experiment with 10% NaCl	-
				Indoles	-	Growth in pH4 Medium	-
Hydrolysis of casein	+	Growth in pH6 Medium	+
				Hydrolysis of tyrosine	-	Growth in pH10 Medium	+
Growth at 5 deg.C	-	Growth in pH11 Medium	-
				Growth at 10 deg.C	-	Urease	+
Growth at 20 deg.C	+	Utilization of malonic acid salt	-
				Growth at 30 ℃	+	Hydrogen sulfide test	-
Growth at 40 deg.C	+	Ornithine dehydroxygenases	-
				Growth at 42 deg.C	-	Arginine dehydroxylase	-
Arginine double hydrolase	-

+: positive; -: negative of

Molecular biological identification: extracting the total DNA of the strain by using a total DNA extraction kit, and performing PCR amplification and sequencing (George et al, 2013) on a genome of the strain by using 16S rDNA universal primers F27 and R1492, wherein the sequence is shown as SEQ ID NO: 1 is shown. And (3) carrying out homology comparison on the sequencing result and a reported 16S rDNA sequence in GenBank, and carrying out phylogenetic evolutionary tree analysis, wherein the 16S rDNA sequence of the strain YLYPP 1 has higher homology with Bacillus megaterium B2(KT307979.1), the homology rate reaches 99%, and the constructed phylogenetic evolutionary tree is shown in figure 3. Therefore, the strain was identified as bacillus megaterium (bacillus megaterium) based on morphological characteristics, physiological and biochemical characteristics, and phylogenetic analysis of the strain. The strain is named as Bacillus megaterium YLLYP 1.

The preservation information of Bacillus megaterium YLYPP 1: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: day 21, 2017, 06 and 21, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60204.

example 3

The phosphorus dissolving capacity of the strain Bacillus megaterium YLLYP 1 is determined as follows:

(1) preparation of bacterial suspension: selecting a small amount of YLYP1 strain stored on the slant surface of a test tube by using an inoculating loop, culturing for 12h in 50mL LB liquid culture medium at 30 ℃ and 150rpm, taking 40mL of culture solution in a 50mL centrifuge tube, centrifuging for 3min at 4 ℃ and 3500rpm, collecting the strain, washing the strain for 3 times by using 0.9% sterile physiological saline, and preparing the strain with the strain density of 10 by using the physiological saline⁸cfu/mL of bacterial suspension.

(2) Phosphate pretreatment: weighing 20.0g of analytically pure aluminum phosphate, iron phosphate or calcium phosphate in a big beaker, ultrasonically dissolving the analytically pure aluminum phosphate, iron phosphate or calcium phosphate with 2L of ultrapure water, discarding the upper-layer turbid liquid every 30min, keeping the indissolvable aluminum phosphate, iron phosphate or calcium phosphate at the bottom of the beaker, repeatedly ultrasonically washing the indissolvable aluminum phosphate, iron phosphate or calcium phosphate for 2-3 times until the upper-layer turbid liquid is clarified, collecting the aluminum phosphate, iron phosphate or calcium phosphate precipitated at the bottom of the beaker, placing the beaker in a 60 ℃ drying oven for drying, grinding the beaker, placing the beaker in a brown reagent.

(3) Preparation of NBRIP culture medium:

preparing a culture medium by using indissolvable aluminum phosphate, iron phosphate or calcium phosphate as a unique phosphorus source, adjusting the pH value to 7.0 by using a NaOH solution, and sterilizing at 121 ℃ for 20 min. NBRIP medium formulation is shown in step (2) of example 1.

(4) Inoculation and culture: mixing the prepared bacterial suspension (10)⁸cfu/mL) was measured according to 4% (V: v) was inoculated into NBRIP medium. The culture was carried out at 30 ℃ and 130rpm for 5 days.

(5) Determination of soluble phosphate: after culturing for 5d, taking 2mL of culture solution in a centrifuge tube, centrifuging at 4 ℃ and 3500rpm for 10min, taking 0.2mL of supernatant to dilute to 10mL, and determining the content of soluble phosphate by using a molybdenum-antimony anti-spectrophotometry (determination of sodium bicarbonate leaching-molybdenum-antimony anti-spectrophotometry of HJ 704-2014 soil available phosphorus). A suspension of bacteria sterilized at 121 ℃ for 20min was used as a control. And dissolving aluminum phosphate in the NBRIP culture medium to obtain a soluble phosphorus content of 95.3 +/-3.2 mg/L, dissolving iron phosphate to obtain a soluble phosphorus content of 130.4 +/-5.6 mg/L, and dissolving insoluble calcium phosphate to release soluble phosphorus of 405.0 +/-3.7 mg/L. To exclude the effect of autoclaving on the soluble phosphate content of the broth, we measured the content of soluble phosphate in the broth before and after sterilization, and the results showed that autoclaving had no effect on the soluble phosphate content.

Example 4

And (3) analyzing the phosphorus dissolving capacity of the complex microbial inoculum:

(1) inoculating Bacillus megaterium YLLYP 1 and Rhodococcus pyridinivorans XB into LB liquid culture medium, respectively, and activating at 30 deg.C for 12 h;

(2) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10⁹cfu/mL；

(3) The 2 single strains are treated according to the following steps of 2: 1, and then evenly mixing to obtain the multifunctional complex microbial inoculum.

(4) The complex microbial inoculum, Bacillus megaterium YLLYP 1 and Rhodococcus pyrivorans XB with equal amount of thallus are respectively inoculated into NBRIP culture medium (the only phosphorus source adopts calcium phosphate), and cultured at 30 deg.C and 130rpm for 5 days, with 3 times of treatment setting.

(5) Determination of soluble phosphate: after culturing for 5 days, 2mL of culture solution is taken to be put in a centrifuge tube, centrifugation is carried out at 4 ℃ and 3500rpm for 10min, 0.2mL of supernatant is taken to be diluted to 10mL, and the content of soluble phosphate is determined by a molybdenum-antimony anti-spectrophotometry method. The result shows that the content of soluble phosphorus in the culture solution of Bacillus megaterium YLLYP 1 is 413.2mg/L, the content of soluble phosphorus in the Rhodococcus pyridinivorans XB culture solution is 18.2mg/L, and the content of soluble phosphorus in the culture solution of the composite microbial inoculum is 529.5mg/L, which indicates that the phosphorus dissolving capacity of the composite microbial inoculum is obviously improved, and the compounding effect of 1+1>2 microbial inoculum is achieved.

Example 5

(1) inoculating Bacillus megaterium YLLYP 1 and Rhodococcus pyridinivorans XB into LB liquid culture medium, respectively, and activating at 28 deg.C for 12 h;

(2) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10⁸cfu/mL；

(3) The 2 single strains are treated according to the following steps of 2: 3, and evenly mixing the mixture according to the volume ratio to obtain the multifunctional composite microbial inoculum.

(5) Determination of soluble phosphate: after culturing for 5 days, 2mL of culture solution is taken to be put in a centrifuge tube, centrifugation is carried out at 4 ℃ and 3500rpm for 10min, 0.2mL of supernatant is taken to be diluted to 10mL, and the content of soluble phosphate is determined by a molybdenum-antimony anti-spectrophotometry method. The result shows that the soluble phosphorus content in the culture solution of Bacillus megaterium YLLYP 1 is 405.0mg/L, the soluble phosphorus content in the Rhodococcus pyridinivorans XB culture solution is 15.7mg/L, and the soluble phosphorus content in the culture solution of the composite microbial inoculum is 482.5mg/L, which indicates that the phosphorus dissolving capacity of the composite microbial inoculum is obviously improved, and the compounding effect of 1+1>2 microbial inoculum is achieved.

Example 6

(1) inoculating Bacillus megaterium YLLYP 1 and Rhodococcus pyridinivorans XB into LB liquid culture medium, respectively, and activating at 30 deg.C for 18 h;

(2) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10¹⁰cfu/mL；

(3) The 2 single strains are treated according to the following steps of 2: 2, and then evenly mixing the mixture to obtain the multifunctional composite microbial inoculum.

(5) Determination of soluble phosphate: after culturing for 5 days, 2mL of culture solution is taken and put in a centrifuge tube, centrifugation is carried out at 4 ℃ and 3500rpm for 10min, 0.2mL of supernatant is taken and diluted to 10mL, and the content of soluble phosphate is determined by a molybdenum-antimony anti-spectrophotometry method. The result shows that the soluble phosphorus content in the Bacillus megaterium YLLYP 1 culture solution is 436.2mg/L, the soluble phosphorus content in the Rhodococcus pyridinivorans XB culture solution is 20.2mg/L, and the soluble phosphorus content in the composite microbial inoculum culture solution is 570.3mg/L, which indicates that the phosphorus dissolving capacity of the composite microbial inoculum is remarkably improved, and the compounding effect of 1+1>2 microbial inoculum is achieved.

Example 7

Analysis of the capacity of the compound microbial inoculum for degrading DEHP:

(4) Respectively inoculating Bacillus megaterium YLLYP 1 and Rhodococcus pyrivorans XB with equal thallus amount and composite microbial inoculum into MSM culture medium with DEHP as the only carbon source, culturing at 30 deg.C and 130rpm for 3 days, and setting 3 times of treatment with non-bacterial culture medium as blank control.

(5) DEHP in the culture solution of Bacillus megaterium YLVP 1 is not degraded, the degradation rate of the Rhodococcus dipyridinivorans XB on the DEHP is 80.5%, and the degradation rate of the compound microbial inoculum on the DEHP is 90.5%. Therefore, the degradation capability of the compound microbial inoculum on DEHP is improved.

Example 8

Analysis of the capacity of the compound microbial inoculum for degrading DEHP:

(4) Respectively inoculating single bacteria Bacillus megaterium YLVP 1, Rhodococcus pyrivorans XB and composite microbial inoculum with equal amount of bacteria into MSM culture medium with DEHP as the only carbon source, culturing for 3 days in a sterile shaking table at 30 ℃ and 150rpm, and setting 3 times of treatment by using a non-bacterial culture medium as a blank control.

(5) DEHP in the culture solution of Bacillus megaterium YLVP 1 is not degraded, the degradation rate of DEHP by Rhodococcus pyritivivorans XB is 91.9%, and DEHP is completely degraded by the complex microbial inoculum. Therefore, the degradation capability of the compound microbial inoculum on DEHP is improved.

Example 9

The composite microbial inoculum is applied to the improvement of phosphorus-deficient soil:

(1) test soil: the pH value of the farmland paddy soil is 5.62, the organic matter content is 17.40g/kg, the total nitrogen content is 0.79g/kg, the total phosphorus content is 0.66g/kg, and the farmland paddy soil is dried by air and then passes through a 60-mesh sieve.

(2) Inoculating Bacillus megaterium YLLYP 1 and Rhodococcus pyridinivorans XB into LB liquid culture medium, respectively, and activating at 30 deg.C for 18 h;

(3) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10⁸cfu/mL；

(4) The 2 single strains are treated according to the following steps of 2: 1, and then evenly mixing to obtain the multifunctional complex microbial inoculum.

(5) And (3) measuring the content of available phosphorus in soil:

adding 600g of soil into a 500mL beaker, shading with tinfoil paper, adding 60mL of the bacterial strains YLVP 1, XB and the complex microbial inoculum into the soil respectively, and uniformly mixing. Adjusting the water content of the soil to 30% of the field water capacity, culturing at 30 ℃ for 10d, and setting three times for each treatment by taking the soil without any microbial inoculum as a reference. The effective phosphorus content in the soil treated by different methods is measured, and the results show that the effective phosphorus content in the contrast is 39.8mg/kg, the effective phosphorus content in the soil inoculated with single Bacillus megaterium YLLYP 1 is 59.5mg/kg, the effective phosphorus content in the soil inoculated with single Bacillus Rhodococcus pyritivorans XB is 41.7mg/kg, and the effective phosphorus content in the soil inoculated with the composite microbial inoculum is 76.2 mg/kg.

Example 10

(3) selecting activated 2 single bacteria, respectively inoculating into LB liquid culture medium, respectively culturing until the number of bacteria is 10¹⁰cfu/mL；

(5) And (3) measuring the content of available phosphorus in soil:

adding 600g of soil into a 500mL beaker, shading with tinfoil paper, adding 60mL of the bacterial strains YLVP 1, XB and the complex microbial inoculum into the soil respectively, and uniformly mixing. Adjusting the water content of the soil to 30% of the field water capacity, culturing at 30 ℃ for 10d, and setting three times for each treatment by taking the soil without any microbial inoculum as a reference. The effective phosphorus content in the soil treated by different methods is measured, and the results show that the effective phosphorus content in the contrast is 39.8mg/kg, the effective phosphorus content in the soil inoculated with single Bacillus megaterium YLLYP 1 is 68.1mg/kg, the effective phosphorus content in the soil inoculated with single Bacillus Rhodococcus pyritivorans XB is 45.2mg/kg, and the effective phosphorus content in the soil inoculated with the composite microbial inoculum is 87.2 mg/kg.

Example 11

The compound microbial inoculum is applied to the restoration of DEHP contaminated soil:

(4) The 2 single strains are treated according to the following steps of 2: 3, and evenly mixing the mixture according to the volume ratio to obtain the multifunctional composite microbial inoculum.

(5) Repairing DEHP contaminated soil by using composite microbial inoculum

The DEHP contamination concentration was set at 20 mg/kg. Adding 600g of soil into a 500mL beaker, adding DEHP, stirring uniformly, shading with tinfoil paper, aging for one week, adding 60mL of the bacterial strains YLYP1, XB and the composite microbial inoculum into the soil, and stirring uniformly. Adjusting the water content of the soil to be 30% of the field water capacity, culturing at the constant temperature of 30 ℃ for 15d, taking the soil without any microbial inoculum as a blank control, setting 3 times of repetition for each treatment, and measuring the residual quantity of DEHP in the process. The results showed that the soil degradation rate of DEHP in the blank control was 27.1%, the soil inoculated with Bacillus megaterium YLVP 1 was 32.2%, the soil inoculated with Bacillus sclerotivorans XB was 53.9%, and the soil added with the complex microbial inoculum had a DEHP degradation rate of 70.3%.

Example 12

(5) Repairing DEHP contaminated soil by using composite microbial inoculum

The DEHP contamination concentration was set at 20 mg/kg. Adding 600g of soil into a 500mL beaker, adding DEHP, stirring uniformly, shading with tinfoil paper, aging for one week, adding 60mL of the bacterial strains YLYP1, XB and the composite microbial inoculum into the soil, and stirring uniformly. Adjusting the water content of the soil to be 30% of the field water capacity, culturing at the constant temperature of 30 ℃ for 15d, taking the soil without any microbial inoculum as a blank control, setting 3 times of repetition for each treatment, and measuring the residual quantity of DEHP in the process. The results show that the degradation rate of DEHP in the soil in the blank control is 27.1%, the degradation rate of DEHP in the soil inoculated with the single bacterium Bacillus YLYPP 1 is 36.5%, the degradation rate of DEHP in the soil inoculated with the single bacterium Rhodococcus pyritivorans XB is 65.3%, and the degradation rate of DEHP in the soil added with the composite microbial inoculum is 93.7% respectively.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> phosphate solubilizing bacterium, composite microbial inoculum of phosphate solubilizing bacterium and DEHP degrading bacterium and application of phosphate solubilizing bacterium and DEHP degrading bacterium in soil improvement

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1450

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 16S rDNA sequence of Bacillus megaterium YLLYP 1

<400>1

agcctggggc tctataatgc agtcgagcga ctgattagaa gcttgcttct atgacgttag 60

cggcggacgg gtgagtaaca cgtgggcaac ctgcctgtaa gactgggata acttcgggaa 120

accgaagcta ataccggata ggatcttctc cttcatggga gatgattgaa agatggtttc 180

ggctatcact tacagatggg cccgcggtgc attagctagt tggtgaggta acggctcacc 240

aaggcaacga tgcatagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggctttc gggtcgtaaa actctgttgt tagggaagaa 420

caagtacgag agtaactgct cgtaccttga cggtacctaa ccagaaagcc acggctaact 480

acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggaatt attgggcgta 540

aagcgcgcgc aggcggtttc ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg 600

tcattggaaa ctggggaact tgagtgcaga agagaaaagc ggaattccac gtgtagcggt 660

gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc ggctttttgg tctgtaactg 720

acgctgaggc gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg 780

taaacgatga gtgctaagtg ttagagggtt tccgcccttt agtgctgcag ctaacgcatt 840

aagcactccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc 900

cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 960

tgacatcctc tgacaactct agagatagag cgttcccctt cgggggacag agtgacaggt 1020

ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080

aacccttgat cttagttgcc agcatttagt tgggcactct aaggtgactg ccggtgacaa 1140

accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200

gtgctacaat ggatggtaca aagggctgca agaccgcgag gtcaagccaa tcccataaaa 1260

ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagctggaa tcgctagtaa 1320

tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380

ccacgagagt ttgtaacacc cgaagtcggt ggagtaaccg taaggagcta gccgcgtaag 1440

gtgaacaaat 1450

Claims

1. A phosphorus-solubilizing bacterium is characterized in that: name isBacillus megateriumYLYP1, deposited at 21.06.7.7 in the Guangzhou province, Guangzhou, Minichuo No. 100 college No. 59 building, Guangdong province, microbiological research institute, accession number: GDMCC NO: 60204.

2. use of the phosphorus-solubilizing bacterium according to claim 1 for solubilizing a sparingly soluble phosphate.

3. Use according to claim 2, characterized in that:

4. A complex microbial inoculum is characterized in that: comprising the compound of claim 1Bacillus megateriumYLYP1 andRhodococcus pyridinivoransXB；

saidRhodococcus pyridinivoransXB, deposited in Guangdong provincial microorganism culture collection of Guangdong provincial institute of microbiology, No. 59 building, No. 5 building, Middlexue No. 100 college, Middlexue, Guangzhou, No. 09 and 28 days, 2016, with the collection number: GDMCCNO: 60054, respectively;

saidBacillus megateriumYLYP1 andRhodococcus pyridinivoransXB is respectively made to have a concentration of 10⁸～10¹⁰cfu/mL of bacterial suspension;

saidBacillus megateriumYLYP1 andRhodococcus pyridinivoransXB is added according to the volume ratio of the bacterial suspension 2: 1-3, and mixing.

5. The method for preparing a complex microbial inoculum of claim 4, which is characterized by comprising the following steps:

(1) will be provided withBacillus megateriumYLYP1 andRhodococcus pyridinivoransrespectively inoculating XB into an LB liquid culture medium, and activating for 12-18 h at 28-30 ℃;

(3) The 2 single strains are treated according to the following steps of 2: and (3) uniformly mixing the components in a volume ratio of 1-3 to obtain the multifunctional composite microbial inoculum.

6. The use of the complex microbial inoculum of claim 4 in dissolving phosphorus and/or degrading diisooctyl phthalate.

7. The use of the complex microbial inoculum of claim 4 in improving soil quality.