CN113333459B

CN113333459B - Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same

Info

Publication number: CN113333459B
Application number: CN202110634722.2A
Authority: CN
Inventors: 田江; 郝少芬; 唐斐; 葛飞; 李峰; 岳嘉如
Original assignee: Xiangtan University
Current assignee: Xiangtan University
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2022-06-03
Anticipated expiration: 2041-06-08
Also published as: CN113333459A

Abstract

The invention discloses solid fermentation of penicillium oxalicum, wherein the penicillium oxalicum PSF-4 is used for degrading phosphorus and lead. The solid fermentation of the penicillium oxalicum and the efficient phosphate-dissolving and lead-removing method can realize the effect of dissolving phosphate and removing heavy metals, have low cost, and can improve the soil fertility and the ecological toxicity in the phosphorus-deficient soil seriously polluted by the heavy metals.

Description

Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same

Technical Field

The invention relates to the technical field of heavy metal contaminated soil remediation, in particular to a solid fermentation method of penicillium oxalicum and a method for efficiently dissolving phosphorus and removing lead by using the same.

Background

Due to the aggravation of human activities, the heavy metal pollution of the soil in China is very serious. Heavy metal pollution seriously threatens human health and the stability of an ecological system due to the characteristics of high toxicity, difficult degradability, biological enrichment and the like. Lead is one of the most common toxic heavy metals, belongs to 2B carcinogens, has certain teratogenicity and mutagenicity, and can generate hidden irreversible damage to human bodies due to lead poisoning.

In addition, more than 95% of phosphorus in soil in China mainly exists in the form of insoluble phosphate, and cannot be directly absorbed and utilized by crops, so that the growth and development of the crops are severely restricted. At present, phosphorus is provided for crops by human beings by applying phosphate fertilizers, and the fertilizers often contain heavy metals such as lead and cadmium, so that heavy metal pollution is further aggravated, and meanwhile, the agricultural production cost is increased.

With the development of technology, microbial-based bioremediation technology has attracted great interest to scientists due to its outstanding advantages of high efficiency, low cost, environmental protection, etc. Compared with animals and plants, microorganisms are diverse in variety and extremely widely distributed, and microorganisms can resist environmental stress through rapid mutation and evolution. Therefore, in the environment polluted by the toxic heavy metal ions, the microorganisms can overcome the toxic action of the toxic heavy metal ions by directed evolution and establishment of a resistance system. In addition, some microorganisms can secrete special metabolites through self metabolism, so that active heavy metal ions are converted into an inactive form to repair the heavy metal ions, and bioremediation of a polluted area is realized.

Disclosure of Invention

The invention aims to provide a solid fermentation method of penicillium oxalicum and a method for efficiently dissolving phosphorus and removing lead by the solid fermentation method, which can simultaneously realize the effects of dissolving phosphorus and removing heavy metals, have low cost and can improve the soil fertility and the ecological toxicity in phosphorus-deficient soil seriously polluted by heavy metals.

In order to achieve the purpose, the invention provides solid fermentation of penicillium oxalicum, wherein the solid fermentation of the penicillium oxalicum PSF-4 is used for degrading phosphorus and lead.

A method for efficiently dissolving phosphorus and removing lead by solid fermentation of penicillium oxalicum comprises the following steps:

s1, inoculating the penicillium oxalicum PSF-4 into the fermentation product for fermentation for 3-5 days;

s2, diluting the PSF-4 spores of the penicillium oxalicum produced by solid fermentation to 10⁷And (3) inoculating the CFU/mL into a medium to be decomposed according to the proportion of 1% (v/v) for culture, and repairing at the temperature of 28 ℃ for 7 days.

Preferably, in step S1, the fermentation product is corn flour, peanut straw or fungal solid PDA medium.

Preferably, Pb²⁺The minimum inhibitory concentration on the solid fermentation of the penicillium oxalicum PSF-4 is 1000 mg/L.

Preferably, the solid fermentation of Penicillium oxalicum PSF-4 is carried out on Pb²⁺The removal rate of the phosphorus removal agent is more than 99 percent, and the phosphorus dissolution rate is more than 89 percent on the 4 th to 6 th days of repair.

Therefore, the solid fermentation of the penicillium oxalicum and the efficient phosphate-dissolving and lead-removing method thereof are adopted, the environment-friendly microbial remediation technology is used, phosphate-dissolving bacteria widely existing in the environment are utilized, waste biomass in agricultural production is used as a raw material, the solid fermentation effect of microorganisms is realized, and the phosphate-dissolving capacity and Pb are improved simultaneously²⁺The removal effect is achieved, and therefore the purposes of efficiently removing phosphorus and heavy metals are achieved. The solid fermentation method of the invention has the advantages of low cost and fermentationThe method has the advantages of high speed, large generated microbial thalli and the like, and the obtained mass of phosphate-solubilizing fungal thalli has good application value in the aspects of improving the phosphorus element of farmland soil, improving the soil fertility, promoting the comprehensive treatment of heavy metal soil pollution and the like.

The solid fermented penicillium oxalicum PSF-4 achieves the effect of efficiently dissolving phosphorus by secreting organic acid to dissolve calcium phosphate; by means of FTIR and XRD characterization, the penicillium oxalicum PSF-4 can solidify Pb by secreting organic acid to dissolve calcium phosphate²⁺And stable secondary minerals are generated, and lead ions can be adsorbed on the surface of the mycelium, so that the effect of removing lead is achieved.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 shows the sporulation of the phosphate-solubilizing fungi of corn flour (A), peanut straw (B) and PDA (C) after 4 days of fermentation;

FIG. 2 shows the sporulation yield of the phosphate-solubilizing fungi of corn flour, peanut straw and PDA after 4 days of fermentation;

FIG. 3 shows that PSF-4 is at different Pb²⁺Colony morphology at day 14 in PDA medium concentration;

FIG. 4 shows different initial Pb²⁺Pb at a concentration of (100, 500, 100mg/L)²⁺Change in concentration (A) and amount of dissolved phosphorus (B); pb at different initial pH (3, 6.8, 9) values²⁺Change in concentration (C) and amount of dissolved phosphorus (D);

FIG. 5 shows different treatment groups (Pb)²⁺: 0. 100, 500, 1000mg/L) PSF-4 mycelium surface FTIR spectra;

FIG. 6 shows different treatment groups (Pb)²⁺: 100. 500mg/L) XRD pattern of the precipitate.

Detailed Description

The technical solution of the present invention is further illustrated by the accompanying drawings and examples.

Example one

Solid fermentation of biomass

Scraping appropriate amount of mycelium on the solid plate with inoculating loop, adding 20mL sterile water, shaking thoroughly, sucking appropriate amount of bacterial suspension, and counting with blood cellsPlates were counted and the spore concentration diluted to 10 with sterile water⁷CFU/mL, to prepare an inoculum for later use.

Weighing 10g of dried corn flour and 10g of peanut straw respectively, filling into a dry 250mL conical flask, repeating each group for 3 times, setting a blank control without inoculating PSF-4, subpackaging, and sterilizing in a high-pressure steam sterilizing pot at 121 ℃ for 30 min. After sterilization, 1mL of each inoculum solution was added to give a medium with a spore concentration of 10⁶CFU/g (blank was replaced with 1mL sterile water); and adding 20mL of sterile water, uniformly stirring by using a sterilized glass rod to enable the solid matrix to be blocky (stirring a blank group firstly, using different glass rods for different matrixes), placing the solid matrix in a constant-temperature incubator for culture at 30 ℃ after inoculation is finished, turning over once every 24 hours, and adding 1mL of sterile water every 1d to supplement water lost by evaporation.

As shown in FIG. 1, after 4d of culture, a large amount of hyphae grew on both solid substrates, 100mL of sterile water was added thereto, 5-6 glass beads were added thereto, the mixture was shaken for 30s with a shaking mixer to drop the solid substrate attached to the inner wall of the flask, the mixture was placed in a shaker at 180rpm for 1h, and after shaking was completed, 1mL of bacterial solution was aspirated to dilute the mixture by 10 times, and the count was performed with a hemocytometer. Through calculation, the amount of penicillium oxalicum spores generated by corn flour and peanut straw respectively reaches 3.28 multiplied by 10⁹CFU/g and 2.42X 10⁹CFU/g (FIG. 2), is the initial inoculation fungal spore amount (10)⁶CFU/g) of the sample.

Example two

Fermentation of traditional fungal culture media

Weighing 10g of potato dextrose agar (PDA, a commercial product of solid fermentation in laboratories) which is a traditional fungus culture medium in laboratories, placing the weighed materials into a 250mL conical flask, adding 100mL of water for dissolving, and sterilizing the materials for 30min at 121 ℃ in an autoclave. After sterilization, the solidified medium was triturated with a red-fired cooled inoculating loop and 1mL of each inoculating liquid was added to give a medium having an initial spore concentration of 10 spores as in example one⁶CFU/g (blank was replaced with 1mL sterile water), inoculated and inverted at 30 ℃ in a thermostatted incubator every 24h and 1mL sterile water was added.

As shown in FIG. 1, P was cultured for 4dHypha grows on the DA solid matrix, 100mL of sterile water is added into the DA solid matrix, 5-6 glass beads are added into the DA solid matrix, the DA solid matrix is vibrated for 30s by using a vibration mixer to enable the solid matrix attached to the inner wall of the conical flask to fall off, then the DA solid matrix is placed in a shaking table to be vibrated for 1h at 180rpm, 1mL of bacterial liquid is absorbed after the vibration is finished to be diluted by 10 times, and a blood counting plate is used for counting. The calculated amount of the produced penicillium oxalicum spores respectively reaches 2.23 multiplied by 10⁹CFU/g (FIG. 2), is the initial inoculation fungal spore amount (10)⁶CFU/g) of the sample. But lower than the sporulation yield of corn meal and peanut straw in example one.

The price of PDA culture medium sold in the market is 200 yuan/250 g, and the price of corn flour and peanut straw is <50 yuan/1000 g (network reference price). Therefore, according to the spore-forming concentration and the cost of the two solid fermentations, the corn flour and the peanut straw can be used as good fungus solid fermentations for commercialization and popularization.

EXAMPLE III

Pb²⁺Minimum inhibitory concentration of

Diluting Penicillium oxalicum PSF-4 spores generated by fermenting corn flour and peanut straws to 10 degrees by using sterile water⁷CFU/mL, and 1uL spore suspension inoculated into the center of PDA culture dish. Experimental setup blank and treatment groups (Pb)²⁺: 0. 150, 250, 350, 750 and 1000mg/L) in each treatment, 3 times, observing the growth of colonies after culturing at the constant temperature of 28 ℃ for 2 weeks, and taking the concentration for completely inhibiting the growth of the strain as Pb²⁺Minimal inhibitory concentration against PSF-4.

FIG. 3 shows that the Penicillium oxalicum PSF-4 is Pb-free²⁺The bacterial colony in the medium is dark green, and Pb is added²⁺The post-colony becomes rosy red with Pb²⁺The diameter of the colony becomes smaller as the concentration increases, when Pb²⁺At a concentration of 1000mg/L, no significant colony formation was observed in the culture dish. Therefore, 1000mg/L was determined to be Pb²⁺Minimum inhibitory concentration against PSF-4.

Example four

Different initial Pb²⁺Phosphate solubilizing ability and Pb at concentration²⁺Removal rate verification

PDL (ingredient: potato extract powder 6g/L,glucose 20g/L, agar 20g/L, pH 6.8) medium and phosphate-dissolving medium (PDL + Pi: addition of Ca to PDL Medium₃(PO₄)₂And keeping the volume to 5g/L), and sterilizing in an autoclave for later use.

Diluting Penicillium oxalicum PSF-4 spores generated by fermenting corn flour and peanut straws to 10 degrees by using sterile water⁷CFU/mL, at a rate of 1% (v/v), was added to a 250mL Erlenmeyer flask containing 100mL PDL for two days for preculture. The mycelium pellet was then washed twice with sterile water, blotted dry with sterile filter paper, transferred to a 250ml Erlenmeyer flask containing 100ml of PDL + Pi, Pb²⁺Initial concentrations were set to 0, 100, 500, 1000mg/L, and Pb was added²⁺PDL medium at a concentration of 500mg/L plus PSF-4 served as a control. Continuously culturing for 7 days, and detecting the phosphorus dissolving amount and Pb of the supernatant every 24h²⁺And (4) concentration.

And detecting the content of soluble phosphorus in the supernatant by using a phosphorus-antimony-molybdenum blue spectrophotometry. By using KH₂PO₄The phosphorus standard curve is drawn, and the correlation coefficient of linear fitting reaches 0.9991. When the soluble phosphorus content of the supernatant is detected, a pipette is used to accurately suck 50 mu L of the supernatant every day, the supernatant is added into a centrifuge tube filled with 950 mu L of distilled water, then the centrifuge tube is centrifuged at 12000rpm for 1min, the supernatant is transferred into a 25mL colorimetric tube, 1mL of 10% ascorbic acid solution is added into the colorimetric tube, and the mixture is uniformly mixed. And adding 2mL of molybdate solution after 30s, fully mixing, standing for 15min, adding into a cuvette at the wavelength of 700nm, measuring the absorbance, taking a zero-concentration solution as a reference, and finally calculating the content of soluble phosphorus according to a standard curve value.

Detection of Pb in supernatant Using flame atomic absorption²⁺And (4) concentration. Using Pb (NO)₃)₂And drawing a lead standard curve, and linearly fitting the correlation coefficient to 0.9996. Detecting Pb in the supernatant²⁺When the concentration is required, a pipette is used to accurately suck 2mL of supernatant every day, the supernatant is centrifuged and filtered through a 0.45um filter membrane, then the supernatant is diluted to a proper concentration by 0.2% nitric acid, and the concentration is measured by an atomic absorption spectrophotometer.

EXAMPLE five

Phosphate solubilizing ability and Pb at different initial pH values²⁺Removal rate verification

Adjusting initial pH value in PDL medium to 3, 6.8 and 9 by using 1mol/L NaOH and HCL, wherein 6.8 is the original pH value of the medium, and taking Ca₃(PO₄)₂Subpackaging, and sterilizing at 121 deg.C for 20 min. Preparation of Pb (NO)₃)₂The mother liquor is filtered through a 0.22um filter membrane and then mixed with Ca₃(PO₄)₂Adding into sterilized PDL medium to make the culture medium contain Pb²⁺And Ca₃(PO₄)₂The concentration was 500mg/L and 5g/L, respectively.

After harvesting fresh spores, as in example three, they were diluted to 10⁷one/mL, and inoculated at 1% into a 250mL Erlenmeyer flask containing 100mL PDL for 2 days of pre-culture, transferred to a medium of PDL + Pi of different initial pH values, with PDL + Pi of pH 6.8 as a control without addition of bacteria and PDL + PSF-4. Continuously culturing for 7 days, and detecting the phosphorus dissolving amount and Pb of the culture medium supernatant every 24h²⁺The concentration and detection method are the same as in example two.

FIG. 4 shows the results of experiments with simultaneous addition of Ca to PDL medium₃(PO₄)₂And PSF-4 can be at a higher initial Pb²⁺Realizes efficient phosphate dissolution and Pb removal in the concentration and large pH value change range²⁺The effect of (1). Pb²⁺The removal rate of the phosphorus removal agent is over 99 percent, and the phosphorus dissolution amount is basically over 900 mg/L. At Pb²⁺In PDL + Pi culture media with initial concentrations of 100, 500 and 1000mg/L, the removal rates of the first day respectively reach 99.6%, 99.5% and 99.9%; the maximum phosphorus dissolving amount is reached in 4-6 days, the phosphorus dissolving amount is 934, 956 and 1000mg/L respectively, and the phosphorus dissolving rate is 93.4%, 95.6% and 100% respectively. Pb in PDL + Pi medium at initial pH values of 3, 6.8, and 9 on days 1-2²⁺The maximum removal rate is respectively 99.9 percent, 99.5 percent and 99.5 percent; the maximum phosphorus dissolving amount is achieved in 4-6 days, which is 947, 956 and 896mg/L respectively, and the phosphorus dissolving rate is 94.7%, 95.6% and 89.6% respectively. Although Pb in the solution at the late stage of the cultivation²⁺The concentration is increased, but the removal rate is always kept above 98 percent, which indicates that PSF-4 is applied to Pb²⁺The removal of (2) has higher stability.

Example six

Pb²⁺To phosphate solubilizing myceliumAnalysis of the influence of surface functional groups

Different initial Pb of example III²⁺Mycelia in a PDL + Pi medium were washed three times with sterile water, dried in a vacuum oven at 60 ℃ to a constant weight, and the dried mycelia were pulverized. Taking a proper amount of powder, mixing a mycelium sample with potassium bromide according to the ratio of 1: 50, mixing, tabletting and detecting by FTIR.

The results are shown in FIG. 5, where the mycelium was at Pb²⁺The absorption peak of part of the surface functional groups in the solution changes. 3343 the absorption peak of ammonia compound has-OH and-NH stretching vibration, and the peak shifts in lead solution with different concentrations, and the absorption peak strength increases and widens obviously with the increase of the concentration, which shows that the amino or carboxyl in carbohydrate, cell wall material and protein has ion exchange or complexation with lead. At 2925, the intensity of the absorption peak increases gradually with the increase in the concentration of heavy metals, and the position of the absorption peak shifts to a small extent, mainly due to the C-H stretching vibration in proteins, saccharides, and the like. Absorption peaks around 1641 and 1383 are absorption bands I and II of the amide compound, and the absorption peaks are obviously enhanced in intensity with the increase of the lead concentration, which indicates that amino acids in the protein participate in the absorption of lead. The absorption peak at 1027 is a stretching vibration peak of C-H, C-O, C-C, P-O and the like in carbohydrate. It can be seen that PSF-4 adsorbs Pb through amino, carboxyl, amide, etc. groups on the surface²⁺Is that it removes Pb²⁺One of the ways of (1).

EXAMPLE seven

Phosphate solubilizing bacteria for driving Pb²⁺XRD analysis of the immobilized product

Diluting Penicillium oxalicum PSF-4 spores generated by fermenting corn flour and peanut straws to 10⁷CFU/mL, and 1% inoculation into 250mL Erlenmeyer flasks containing 100mL PDL for 2 days before transfer to the initial Pb²⁺In PDL medium (containing no bacteria and no calcium phosphate as control groups) at a concentration of 1000mg/L, the precipitate in the culture solution on the fourth day was lyophilized, and the precipitated compound was analyzed by X-ray diffraction powder analyzer in the form of powder. Adopting MDI jade 5.0 software to mapAnd performing PDF card comparison to generate the material composition.

As shown in FIG. 6, stable phosphorus-chloroplatinic acid was formed in the medium with the simultaneous addition of Penicillium oxalicum and calcium phosphate. The research of the related literature indicates that the organic acid secreted by the microorganism can dissolve the phosphate which is difficult to dissolve. Thus, PSF-4 is precisely responsible for dissolving calcium phosphate by the secretion of oxalic acid and further for solidifying Pb by the formation of the secondary mineral pnictogen²⁺。

Therefore, the solid fermentation of the penicillium oxalicum and the efficient phosphate-dissolving and lead-removing method thereof can realize the effect of dissolving phosphate and removing heavy metals, have low cost and can improve the soil fertility and the ecological toxicity in the phosphorus-deficient soil seriously polluted by the heavy metals.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims

1. A method for efficiently dissolving phosphorus and removing lead by solid fermentation of penicillium oxalicum is characterized by comprising the following steps: the method comprises the following steps: s1, inoculating the penicillium oxalicum PSF-4 into the fermentation product for fermentation for 3-5 days; s2, diluting the PSF-4 spores of the penicillium oxalicum produced by solid fermentation to 10⁷CFU/mL, inoculating the strain to a culture medium to be decomposed according to the proportion of 1% v/v, culturing, and repairing for 7 days at the temperature of 28 ℃ to obtain the strain; solid fermentation of Penicillium oxalicum PSF-4 on Pb²⁺The removal rate of the composite is more than 99 percent, and the phosphorus dissolution rate is more than 89 percent on the 4 th to 6 th days of repair; pb²⁺The minimum inhibitory concentration on the solid fermentation of the penicillium oxalicum PSF-4 is 1000 mg/L.

2. The method for efficiently decomposing phosphorus and removing lead by solid fermentation of penicillium oxalicum according to claim 1, characterized in that: in step S1, the fermentation product is corn flour, peanut straw or fungus solid PDA culture medium.