CN107475238B - Lotus seed pot immobilized microorganism and preparation method and application thereof - Google Patents
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
The application relates to a preparation method of a lotus seed pot immobilized microorganism, which comprises the following specific steps: (1) preparing a lotus seed pot; (2) immobilizing the microorganisms in the lotus seedpod: a. inoculating WH16-1 bacterial liquid in logarithmic phase into LB culture medium according to the standard of 1% volume fraction, placing in a shaking table for culturing for 12h, centrifuging, removing supernatant, and adding 25mL of fresh culture solution; b. adding receptaculum nelumbinis and sodium alginate into the bacterial liquid, and stirring to mix thoroughly; c. b, injecting the mixed solution obtained in the step b into a calcium chloride solution with a certain mass concentration, standing and solidifying for 6-24 times, pouring out the liquid, cleaning particles, and performing vacuum freeze drying; the lotus seed pot is creatively adopted as the solid carrying material, so that the waste is recycled, and the resources are greatly saved; moreover, the prepared rosette immobilized microorganism has excellent performance on heavy metals, the removal rate of 500mg/L Cr (VI) and 50mg/L Cd (II) in 4 days is 95.32 percent and 80.33 percent respectively, and the lotus seedpot immobilized microorganism has good practical value and application prospect.
Description
Technical Field
The invention belongs to the technical field of materials and environment, and particularly relates to a lotus seed pot immobilized microorganism as well as a preparation method and application thereof.
Background
The lotus seed pot is a mature receptacle of lotus, mainly comprises chemical substances such as polyphenol, alkaloid, protein, saccharide, fat, cellulose, procyanidine, vitamin and the like, and has the effects of resisting oxidation, stopping bleeding, removing blood stasis and eliminating dampness. At present, researches on extraction of procyanidine (Lijiaqiao and the like, 2016) and preparation of crisp biscuits (Lichengjie and the like, 2006) by utilizing the procyanidine have been reported, but actually, with the expansion of the industrial scale of lotus seeds in recent years, the main byproduct lotus seedpod is randomly discarded and burned to cause resource waste and environmental pollution, and resource utilization of the procyanidine is urgently needed to avoid the environmental pollution.
In addition, a large amount of wastewater, waste residues and waste gases containing heavy metals such as chromium and cadmium are generated due to the aggravation of human activities, so that a large amount of heavy metal pollution such as cadmium and chromium in water and soil environments is caused (Viti et al, 2013). Chromium has a common oxidation number of +3 and +6, with cr (vi) being highly soluble, easily migratory, toxic, carcinogenic, teratogenic, mutagenic (Bagchi et al, 2002), cr (iii) being less soluble, unable to cross biological membranes, prone to precipitate with other ions, and less toxic (Francisco et al, 2002), cr (vi) being about 100 times more toxic than cr (iii) (Ehrlich, 2002). Cr (vi) in wastewater is the predominant form present, and thus, conversion of cr (vi) to cr (iii) is a common method for effective removal and reduction of chromium contamination. Soluble cadmium is easily absorbed by plants and then enters a human body through a food chain, and a small amount of cadmium continuously enters the human body to damage tissues and organs, so that obvious lesions are shown in kidneys, livers, lungs, bones, reproductive systems, cardiovascular systems, gastrointestinal systems and pancreas (catkin, 2007). Therefore, the harm of cadmium can be effectively reduced by reducing the content of soluble cadmium. There are many methods for removing chromium and cadmium, and among them, biological methods are receiving attention because of their low investment and running cost and low secondary pollution. The application provides a method for removing heavy metals by using low-cost biomass material lotus seedpod as a carrier to fix microorganisms.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for removing heavy metals by using biomass material lotus seedpod as a carrier to fix microorganisms.
In order to solve the problems, the invention adopts the technical scheme that:
a preparation method of a lotus seed pot immobilized microorganism comprises the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating WH16-1 bacterial liquid in a logarithmic phase into 100mL LB culture medium according to a standard of 1% in volume fraction, then adding 0.5-2 g of lotus seedpod, placing the lotus seedpod in a shaking table at 37 ℃ and 160r/min for culturing for 12h, finally centrifuging, cleaning particles, and carrying out vacuum freeze drying to obtain the lotus seedpod immobilized microorganism particles.
Or the technical scheme of the invention can also be as follows:
a preparation method of a lotus seed pot immobilized microorganism comprises the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating WH16-1 bacterial liquid in logarithmic phase into 100mL LB culture medium according to the volume fraction of 1%, culturing in a shaker at 37 ℃ and 160r/min for 12h, centrifuging, discarding supernatant, and adding 25mL fresh culture solution; b. adding 0.5-2 g of lotus seedpod and 1-3 g of sodium alginate into the bacterial liquid, and stirring to fully mix; c. and c, injecting the mixed solution obtained in the step b into 100g of calcium chloride solution with the mass concentration of 2-4%, standing and solidifying for 6-24 h, pouring out the liquid after backward pouring, washing particulate matters with normal saline, and carrying out vacuum freeze drying to obtain the lotus seed pot immobilized microorganism particles.
In addition, the invention also claims the lotus seed pot immobilized microorganism prepared by the method.
And the application for protecting the lotus seed pot immobilized microorganism is as follows: putting a certain amount of the lotus seed pot immobilized microorganism particles into a culture medium containing a certain amount of Cr (VI) and/or Cd (II), shaking, sampling after a certain time, centrifuging and filtering, measuring the content of the Cr (VI) and Cd (II) in the supernatant, and calculating the removal amount of the lotus seed pot immobilized microorganism to the Cr (VI) and Cd (II) according to the concentration difference of the Cr (VI) and/or Cd (II) before and after treatment.
Compared with the prior art, the rosette immobilized microorganism provided by the invention has the following obvious beneficial effects:
(1) the lotus seed industry by-product lotus seed pot is creatively adopted as the solid carrying material, so that the lotus seed pot is cheap and easy to obtain on one hand, the reutilization of wastes is realized on the other hand, the resources are greatly saved, and the pollution caused by the wastes is avoided;
(2) the lotus seed pot adopted by the invention contains a large amount of cellulose on one hand, is particularly suitable for immobilizing microorganisms, and on the other hand, contains a large amount of nutrient substances such as protein, sugar, fat and the like, provides a very favorable environmental condition for the growth of the microorganisms, and also contains a large amount of reducing substances (antioxidant substances) such as proanthocyanidins with the content of about 7.8 percent, and can reduce Cr (VI), so that the heavy metal can be removed together with the microorganisms;
(3) the sodium alginate solution is added with polyvalent metal ions to be crosslinked into reticular gel; mixing microorganism, receptaculum Nelumbinis and sodium alginate solution, spraying calcium salt solution with needle to obtain beaded gel, and wrapping and fixing microorganism and receptaculum Nelumbinis to form beaded gel for recycling.
(4) The prepared rosehouse immobilized microorganism has excellent removal effect on Cr (VI) and Cd (II) heavy metals, and is far higher than that of free microorganism, wherein the removal rates of 500mg/L Cr (VI) and 50mg/L Cd (II) in 4 days are respectively 95.32% and 80.33%, and no secondary pollution is caused; the method has simple process and good practical value and application prospect.
Drawings
FIG. 1-1 shows the removal results of the lotus seed pot immobilized microorganism and microorganism WH16-1 from 300mg/L Cr (VI) -containing solution in example 1;
FIG. 1-2 shows the results of removing the 500mg/L Cr (VI) -containing solution with the rosette immobilized microorganism and microorganism strain WH16-1 in example 1;
FIG. 2 shows the removal results of the microorganism immobilized in the lotus seed pot, the lotus seed pot and the microorganism WH16-1 in example 2 on a solution containing Cr (VI) at a concentration of 500 mg/L;
FIG. 3 is the result of storage stability of the sodium alginate-immobilized microorganism of lotus seedpod in example 3;
FIG. 4-1 shows the result of removing 9mg/L Cd (II) -containing solution from the lotus seed pot sodium alginate-immobilized microorganism and microorganism WH16-1 in example 4;
FIG. 4-2 shows the removal results of 50mg/L Cd (II) -containing solution from the lotus seed pot sodium alginate-immobilized microorganism and microorganism WH16-1 in example 4;
FIG. 5 shows the result of the removal of Cd (II) by the sodium alginate-immobilized microorganism of receptaculum nelumbinis and microorganism WH16-1 in example 5;
FIG. 6-1 shows the result of removing Cd (II) from the mixed solution of 9mg/LCd (II) and 300mg/L Cr (VI) by using the lotus seed pot sodium alginate-immobilized microorganism and microorganism WH16-1 in example 6;
FIG. 6-2 shows the result of removing Cr (VI) from the mixed solution of 9mg/LCd (II) and 300mg/L Cr (VI) by using the lotus seed pot sodium alginate-immobilized microorganism and microorganism WH16-1 in example 6.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
A preparation method of a lotus seed pot immobilized microorganism comprises the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating a WH16-1 bacterial liquid with a volume fraction of 1% in a growth logarithmic phase into 100mL of LB culture medium, culturing in a shaking table at 37 ℃ and 160r/min for 12h, centrifuging, removing a supernatant, and adding 25mL of fresh culture solution; b. adding 0.5g of the lotus seed pot powder obtained in the step (1) and 25g of sodium alginate solution with the mass fraction of 4% into the liquid obtained in the step a, and stirring to fully and uniformly mix the lotus seed pot powder and the sodium alginate solution; c. and c, injecting the mixed solution obtained in the step b into 100g of calcium chloride solution with the mass concentration of 2%, standing and solidifying for 24 hours, pouring out the liquid after the solution is poured out, washing particles with physiological saline, and carrying out vacuum freeze drying to obtain the lotus seed pot immobilized microorganism particles.
Taking 1.6g of the lotus seed pot immobilized microorganism in 100mL of culture medium containing Cr (VI) (potassium dichromate) with different concentrations, sampling after a certain time interval, centrifugally filtering the sample, and measuring the content of the Cr (VI) remained in the supernatant by using a dibenzoyl Dihydrazide (DPC) spectrophotometry. Three experiments in parallel in each group were performed with the same number of bacteria in 100mL of WH16-1 containing bacterial suspension as a control. And calculating the removal amount of Cr (VI) by the rosette immobilized microorganisms according to the concentration difference of Cr (VI) before and after treatment. The experimental result shows that the removal rates of the free microorganism to 300 and 500mg/L Cr (VI) within 4 days are respectively 80.13 percent and 59.12 percent, and the removal rates of the lotus seed pot sodium alginate-immobilized microorganism to 300 and 500mg/L Cr (VI) within 4 days are respectively 100 percent and 80.48 percent. The results of the experiment are shown in FIG. 1.
Example 2
A preparation method of a lotus seed pot immobilized microorganism comprises the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating a WH16-1 bacterial liquid with a mass fraction of 1% in a growth logarithmic phase into 100mL of LB culture medium, adding 0.5g of lotus seed pot, and culturing in a shaker at 37 ℃ and 160r/min for 12 h; and then centrifuging and vacuum drying to obtain the lotus seed pot immobilized microorganism particles.
The said rosette immobilized microorganism is placed in 100mL culture medium containing 500mg/L Cr (VI) (potassium dichromate), after a certain time interval, sampling is carried out, the sample is centrifugally filtered, and the content of the residual Cr (VI) in the supernatant is measured by the diphenyl carbodihydrazide (DPC) spectrophotometry. The control group is composed of 100mL of WH16-1 bacterial liquid containing the same bacterial count and a single lotus plumule, and each group of experiments are in parallel. And calculating the removal amount of Cr (VI) by the rosette immobilized microorganisms according to the concentration difference of Cr (VI) before and after treatment. The experimental result shows that the removal rate of 500mg/L Cr (VI) by the rosette, the microorganisms and the rosette immobilized microorganisms in 4 days is 48.06%, 59.12% and 95.32%. The results of the experiment are shown in FIG. 2.
Example 3
Experiment for determining storage stability of sodium alginate-immobilized microorganisms in lotus seedpod:
the preparation of the syzygium samarangense sodium alginate immobilized microorganism is the same as that in example 1.
The number of bacteria is the same as 1010cfu/mL of free bacterial powder andafter the lotus seed pot sodium alginate-immobilized microorganism particles are stored in a normal-temperature dry environment for 3 months, 1.6g of lotus seed pot sodium alginate-immobilized microorganism and free bacterium powder containing the same bacterium number are respectively taken and placed in 100mL of LB culture medium, the LB culture medium is placed in a shaking table at 37 ℃ and 160r/min for culture for 12 hours, the bacterium number in the solution is measured by a dilution plate-coating method, and the counting results of the free bacterium powder and the lotus seed pot sodium alginate-immobilized microorganism particles are respectively as follows: 0 and 4X 105cfu/mL。
1.6g (dry weight) of the sodium alginate-immobilized microorganisms of the lotus seedpod which are dried at normal temperature and stored for a certain time are taken to be put in 100mL of culture medium, potassium chromate is added to ensure that the potassium chromate contains 500mg/LCr (VI), a sample is sampled after three days, the sample is centrifuged and filtered, the content of the residual Cr (VI) in the supernatant is measured by a diphenyl carbodihydrazide (DPC) spectrophotometry, and three experiments are designed in parallel. The result shows that the lotus seed pot sodium alginate-immobilized microorganism has good storage stability, and is 82.47%, 80.59%, 76.80% and 73.6% for 500mg/L Cr (VI) after 1, 2, 3 and 4 months of storage respectively. The results of the experiment are shown in FIG. 3.
Example 4
Lotus seedpod sodium alginate immobilized microorganism and Cd removal experiment thereof:
the preparation of the syzygium samarangense sodium alginate immobilized microorganism is the same as that in example 1.
And (3) putting 1.6g (dry weight) of the prepared lotus seed pot sodium alginate immobilized microorganism particles into 100mL of culture medium containing a certain amount of cadmium chloride, shaking at a certain temperature, sampling at intervals, centrifuging and filtering the sample, and taking supernatant to measure the content of cadmium by using an atomic absorption spectrometer. And (3) taking 100mL of WH16-1 bacteria liquid containing the same bacteria number as a control, carrying out three parallel experiments in each group, and calculating the removal amount of Cd (II) by using the lotus seedpod immobilized microorganisms according to the concentration difference of Cd (II) before and after treatment. The result shows that the removal rates of 9mg/L Cd (II) and 50mg/L Cd (II) by free microorganisms within 3 days are 68.43% and 0.5% respectively; the removal rate of 9mg/L and 50mg/L Cd (II) by using the lotus seed pot immobilized microorganism in 4 days is 92.96% and 80.33% respectively. The results of the experiment are shown in FIG. 4.
Example 5
Repeatability determination experiment of lotus seedpod sodium alginate immobilized microorganism:
the preparation of the syzygium samarangense sodium alginate immobilized microorganism is the same as that in example 1.
Taking 1.6g (dry weight) of the lotus seed pot sodium alginate-immobilized microorganisms in 100mL LB culture medium containing 9mg/LCd (II), sampling after three days, centrifuging and filtering the sample, measuring the residual cadmium content and the cadmium removal amount in the supernatant by using an atomic absorption spectrophotometry, then adding a certain amount of cadmium chloride and 20mL of LB culture solution for fresh culture according to the residual cadmium concentration, leading the final concentration of Cd (II) to reach 9mg/L, and taking freeze-dried bacterium powder containing WH16-1 with the same bacterium number as a control, wherein each group of experiments are designed into three parallels. Experimental results show that the removal amount of Cd (II) by the freeze-dried bacterial powder and the lotus seed pot immobilized microorganisms in 6 cycles (18 days) of repeated use is 4.03% and 70.36% respectively. The results of the experiment are shown in FIG. 5.
Example 6
Removing mixed solution of Cd (II) and Cr (VI) by using sodium alginate-immobilized microorganisms in lotus seedpod
The preparation of the syzygium samarangense sodium alginate immobilized microorganism is the same as that in example 1.
0.8g of freeze-dried immobilized beads are taken to be put into 50mL of LB culture solution, certain cadmium chloride and potassium dichromate solution are added to make the final concentrations respectively 9mg/LCd (II) and 300mg/LCr (VI), and the mixture is placed in a shaking table at 37 ℃ and 160 r/min. Sampling is carried out once every certain time, three samples in each group are parallel, and the content of the residual Cd and Cr (VI) in the solution is measured. And using free bacteria with the same bacteria number as a control, and designing three parallels in each group of experiments. The result shows that the removal rate of free microorganisms to 9mg/LCd (II) and 300mg/L Cr (VI) within 80h is 51.68 percent and 65.09 percent respectively; the removal rate of Cd (II) and Cr (VI) in the mixed solution of 9mg/LCd (II) and 300mg/L Cr (VI) by using the rosette immobilized microorganism in 80h is 83.19% and 97.46% respectively. The results of the experiment are shown in FIG. 6.
Finally, it is to be noted that: the above examples do not limit the invention in any way. It will be apparent to those skilled in the art that various modifications and improvements can be made to the present invention. Accordingly, any modification or improvement made without departing from the spirit of the present invention is within the scope of the claimed invention.
Claims (4)
1. A preparation method of a lotus seed pot immobilized microorganism is characterized by comprising the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating WH16-1 bacterial liquid in a logarithmic phase into 100mL LB culture medium according to a standard of 1% in volume fraction, then adding 0.5-2 g of lotus seedpod, placing the lotus seedpod in a shaking table at 37 ℃ and 160r/min for culturing for 12h, finally centrifuging, cleaning particles, and carrying out vacuum freeze drying to obtain the lotus seedpod immobilized microorganism particles.
2. A preparation method of a lotus seed pot immobilized microorganism is characterized by comprising the following steps:
(1) preparing a lotus seed pot: collecting receptaculum Nelumbinis, oven drying at 55 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve to obtain powder;
(2) immobilizing the microorganisms in the lotus seedpod: a. inoculating WH16-1 bacterial liquid in logarithmic phase into 100mL LB culture medium according to the volume fraction of 1%, culturing in a shaker at 37 ℃ and 160r/min for 12h, centrifuging, discarding supernatant, and adding 25mL fresh culture solution; b. adding 0.5-2 g of lotus seedpod and 1-3 g of sodium alginate into the bacterial liquid, and stirring to fully mix; c. and c, injecting the mixed solution obtained in the step b into 100g of calcium chloride solution with the mass concentration of 2-4%, standing and solidifying for 6-24 h, pouring out the liquid after backward pouring, washing particulate matters with normal saline, and carrying out vacuum freeze drying to obtain the lotus seed pot immobilized microorganism particles.
3. A rosette immobilized microorganism prepared by the method of any one of claims 1-2.
4. The application of the rosette immobilized microorganism of claim 3, wherein a certain amount of rosette immobilized microorganism particles are placed in a culture medium containing a certain amount of Cr (VI) and/or Cd (II), shaken, sampled after a certain period of time, centrifugally filtered, the content of Cr (VI) and Cd (II) remaining in supernatant is measured, and the removal amount of the rosette immobilized microorganism to Cr (VI) and Cd (II) is calculated according to the concentration difference of Cr (VI) and/or Cd (II) before and after treatment.
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