CN115710554B - Pleurotus pulmonarius strain for decoloring and removing COD (chemical oxygen demand) in sewage and application thereof - Google Patents
Pleurotus pulmonarius strain for decoloring and removing COD (chemical oxygen demand) in sewage and application thereof Download PDFInfo
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- 241001138370 Pleurotus pulmonarius Species 0.000 title claims abstract description 13
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention discloses a pleurotus pulmonarius strain for decoloring and removing COD (chemical oxygen demand) of sewage and application thereof, belonging to the field of biotechnology, wherein the strain is pleurotus pulmonarius (Pleurotus pulmonis) RG-171 which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at 12 months 08 of 2022; deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation number: cgmccno.40366. The invention takes Phanerochaete chrysosporium as a control, determines the decoloring rate and COD removal rate of each strain on sewage, and then obtains a strain of Pleurotus pulmonarius RG-171 with decoloring and COD removal effects through re-screening. Compared with Phanerochaete chrysosporium which is disclosed in the prior art and is used for treating wastewater, the lung-shaped Pleurotus ostreatus RG-171 has better decoloring and sewage COD degradation effects.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a pleurotus pulmonarius strain for decoloring and removing COD (chemical oxygen demand) in sewage and application thereof.
Background
Fungus degradation was found since the 20 th century, and it was first reported that Phanerochaete chrysosporium (Phanerochaete chrysosporium) was able to secrete lignin degrading enzymes. Ming Tie et al in 1983 demonstrated that they could degrade lignin by means of lignin degrading enzymes or lignin modifying enzymes, and studied that these enzymes belong to extracellular enzymes that can be secreted outside the cell or adsorbed on the cell wall, and that Phanerochaete chrysosporium degraded lignin, cellulose and hemicellulose by the action of these extracellular enzymes. In 1985, bumpus et al found for the first time that Phanerochaete chrysosporium was able to inorganize several hardly degradable organic pollutants by lignin degrading enzymes. Phanerochaete chrysosporium was first adopted by the American department of agriculture forest products laboratory and the fungus decoloration research group of North Carolina State university. In 1982, a special treatment technique was developed, and experimental results show that: when the color of the wastewater is more than 1 ten thousand times, the decoloring is a primary reaction, and when the color of the wastewater is more than 1 ten thousand times, the chromaticity of the wastewater is a zero-order reaction.
Chang et al patented in 1987 for the treatment of pulp and pulping paper waste using Phanerochaete chrysosporium. In 1992 Sublett et al, phanerochaete chrysosporium was immobilized on RBC, and the degradation ability of industrial waste water (red water) of munitions was studied, and experimental results show that under intermittent continuous operation conditions, both simulated red water and true red water can be effectively treated.
The Lewandowsk et al utilized Phanerochaete chrysosporium in 1990 to treat 2-chlorophenol wastewater, introduced the design and operating conditions of different types of reactors, focused on developing PBB and fully mixed reactors. The specific parameters of the reactor design of the reactor for treating harmful wastewater by Phanerochaete chrysosporium are disclosed for the first time. Kapdan et al established a RBC reactor of Coriolus versicolor (Trametes versicolor) in 2002, and studied the biological decolorizing effect of different operating parameters on the biological decolorizing of waste water containing phthalocyanine dyes.
The domestic research starts later on the treatment of industrial wastewater by Phanerochaete chrysosporium, and a degradation reaction system of the Phanerochaete chrysosporium on 3 industrial organic wastewater is established in 2003 Li Huirong and the like. However, there is still a need to obtain a strain with better wastewater treatment effect.
Disclosure of Invention
The invention aims to provide a pleurotus pulmonarius strain for decoloring and removing sewage COD and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a Pleurotus ostreatus (Pleurotus pulmonis) RG-171, wherein the Pleurotus ostreatus RG-171 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12/08 of 2022; deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation number: CGMCC No.40366.
The invention also provides a microbial agent, which comprises the pleurotus pulmonarius RG-171.
The invention also provides an application in treating wetland sewage.
Further, the applications are decolorization and COD degradation.
The invention also provides a method for treating the wetland sewage, which comprises the steps of inoculating the lung-shaped Pleurotus ostreatus RG-171 or the microbial agent into the wetland sewage, and decoloring and degrading COD.
Further, the time for decoloring and COD degradation was 14d.
The invention also provides a wetland sewage treatment agent which comprises the Pleurotus ostreatus RG-171 or the microbial agent.
The invention discloses the following technical effects:
(1) After preliminary screening is carried out by using guaiacol and aniline blue solid culture medium, phanerochaete chrysosporium is used as a reference, the decoloring rate and COD removal rate of the Pleurotus pulmonarius RG-171 on sewage are measured, and the Pleurotus pulmonarius RG-171 is re-screened to obtain the Pleurotus pulmonarius RG-171 with strong decoloring and COD removal capacity. Inoculating lung-shaped Pleurotus ostreatus RG-171 on guaiacol-PDA and aniline blue-PDA solid culture medium, measuring the diameter of the color development ring/color fading ring, and measuring the diameter of the color development ring at 59.37+/-0.74 mm by test measurement on guaiacol-PDA; while the diameter of the discoloured circle was 57.43.+ -. 1.75mm measured on aniline blue-PDA solid medium.
(2) The decolorization rate and COD removal rate of the Pleurotus ostreatus RG-171 in 14d are respectively 3.37% and 116.46% higher than those of the control Phanerochaete chrysosporium.
(3) The lung-shaped Pleurotus ostreatus RG-171 reaches an enzyme peak at 10 days when 20% of wetland sewage is added, the enzyme activity peak is 64.32U/L, the decolorizing rate reaches 74.43% at 14 days, and the COD removing rate reaches 79.85% at 14 days.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a phylogenetic tree of CGMCC40366 and related species thereof constructed based on ITS sequences;
FIG. 2 shows the change in the decolorization rate of the adsorption experiment;
FIG. 3 shows the COD removal rate change in the adsorption experiment;
FIG. 4 shows the laccase activity and the change in the decoloring rate and the COD removal rate during degradation of Pleurotus ostreatus RG-171.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The sewage samples used in the following examples are wetland sewage, and are collected from the national natural protection area wetland park of Xingji lake, the indexes are as follows: the color of the water sample is orange, the color is dark, the turbidity is realized, the pH is 8.3, the chromaticity is 200 times, and the COD is 2375.65 +/-73.15 mg/L.
The composition of each medium used in the following examples:
guaiacol-PDA solid medium: 200g of potato extract, 20g of agar, 20g of glucose, 0.4mL of guaiacol, adding water to a volume of 1L, and sterilizing for 3min at 121 ℃.
Aniline blue-PDA solid medium: 200g of potato extract, 20g of agar, 20g of glucose and 0.1g of aniline blue, adding water to a volume of 1L, and sterilizing for 30min at 121 ℃.
Strain activation medium: glucose 20g, agar 20g, yeast extract 5.0g, KH 2 PO 4 1 g，MgSO 4 ·7H 2 O0.5g，ZnSO 4 ·7H 2 O0.05 g, water is added to a constant volume to 1L, the pH is natural, and the sterilization is carried out for 30min at 121 ℃.
Liquid culture medium containing wetland sewage: sewage water100mL of a sample, 20.0g of glucose, 5.0g of yeast extract powder and KH 2 PO 4 1.0g，MgSO 4 ·7H 2 O 0.5g，ZnSO 4 ·7H 2 O50.0 mg, adding water to a constant volume of 1L, naturally adjusting pH, packaging into 250mL conical flasks, pouring 100mL of liquid culture medium into each flask, and sterilizing at 121deg.C for 30min.
EXAMPLE 1 preliminary screening Using guaiacol and aniline blue solid Medium
24 strains of fungi (including part of edible fungi) collected from northeast areas are respectively inoculated into guaiacol-PDA solid culture medium and aniline blue-PDA solid culture medium, cultured for 7 days at 25 ℃, the hypha diameter, the color development circle diameter and the color fading circle diameter in a culture dish are measured by a cross marking method, the ratio of the hypha diameter to the color development circle and the color fading circle diameter is calculated, and 3 repetitions are set for each strain. Strains with higher laccase or peroxidase activity were selected for subsequent experiments. All data treatments were processed and analyzed using SPSS 24.0 and Microsoft Excel software, with significant differences in overall variance (P < 0.05) as measured by one-way analysis of variance (ANOVA), LSD. The final results are shown in tables 1 and 2. The test shows that the diameter of the color development ring in the guaiacol-PDA solid culture medium of the strain RG-171 is 59.37 plus or minus 0.74mm, and the diameter of the color development ring in the aniline blue-PDA solid culture medium is 57.43 plus or minus 1.75mm.
TABLE 1 color development results of guaiacol plates
Note that: the lower case letters after the same column of values are indicated at P<The difference was significant at the level of 0.05.
TABLE 2 color fading results for aniline blue plates
Note that: the lower case letters after the same column of values are indicated at P<The difference was significant at the level of 0.05.
Example 2 determination of bacterial Strain re-screening of decolorizing Rate and COD removal Rate of Sewage
Adding 20mL of liquid culture medium into a 100mL centrifuge tube, removing 2mL of liquid, adding 2mL of wetland sewage, sterilizing, taking 2 bacterial slices with the diameter of 1cm from a flat plate by using a sterile puncher, inoculating the bacterial slices into the liquid culture medium containing the wetland sewage, and carrying out shaking culture for 15d at the temperature of 25 ℃ at the speed of 150r/min, wherein the wetland sewage accounts for 10% of the total volume of the mixed liquid, and 3 parallel samples are arranged for each treatment. The day of adding the wet land sewage is day 0, COD is measured, and the average value and standard deviation of 3 parallel samples are taken as the final result.
The Phanerochaete chrysosporium (Phanerochaete chrysosporium) CGMCC 5.0776 is used as a control, the 20 strains screened out are used for reactivation and then inoculated into a liquid culture medium containing wetland sewage, the growth condition of the strains in 14d is observed and recorded, the COD value and the sewage chromaticity change condition at 14d are measured, and the sewage COD removal rate and the decoloration rate are calculated. The results are shown in Table 3, and the results show that the COD removal rate of the strain RG-171 reaches 79.85% and is 3.37% higher than that of the control; the decoloring rate reaches 65.89%, is 116.46% higher than that of a control, and has strong decoloring capability.
TABLE 3 removal rate of COD and decoloration Rate of double screened Strain
Note that: the different lower case letters after the same column of values indicate significant differences at the P <0.05 level.
Morphological identification:
the fruiting body is in the form of a fungus cover with the width of 4.5-6.5 cm, the fungus cover is in a fan shape, the mature fruiting body cover edge is cracked into a flap shape, the color is grey or yellow brown, and the surface is smooth; meat quality of the fungus, white to milky; the bacterial folds are prolonged, medium density and unequal length; the length of the stipe is 1.5-2.2 cm, the diameter is 0.7-0.9 cm, the stipe is laterally grown and solid, and the basal part is fluffed. The growth condition of the strain RG-171 on the culture medium is observed, and when the strain RG-171 is inoculated for 5d, the strain can grow on the surface of the culture medium, the growth speed is faster, and hyphae are thicker.
Molecular biology identification:
the phylogenetic tree is constructed based on the ITS sequence by using a maximum likelihood method and a Bayesian method, as shown in figure 1. Phylogenetic tree results show that strain RG-171 has collected on one branch with 2 Pleurotus pulmonis strain from Japan and Sweden, and the support rate ML/BI is 80/0.859. The strain RG-171 was identified as Pleurotus pulmonis in combination with conventional morphology and molecular biology.
And (3) preservation proving:
strain RG-171 was deposited at the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12.08 of 2022; deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation number: cgmccno.40366.
Example 3 experiments on the adsorption of fruiting bodies on wastewater
Sterilizing RG-171 fruiting body stored at 121deg.C for 30min to obtain sterilized fruiting body. The fruit bodies with the same biomass are respectively added into the liquid culture medium filled with the wetland sewage by taking the non-sterilized fruit bodies as a control. Shaking culture at 25deg.C at 150r/min for 14d, and setting 3 parallel samples for each treatment, sampling at 3d, 7d, 10d and 14d respectively for detection on day 0 of adding sewage. COD value and chromaticity were measured, and the average value and standard deviation of 3 replicates were taken as final results. The growth condition of the strain in 14d and the decoloring condition of the sewage culture medium are observed and recorded, the decoloring rate and COD removal rate of the sewage culture medium are calculated, and the average value and standard deviation of 3 parallel samples are taken as final results, and the results are shown in fig. 1 and 2.
Through an adsorption experiment of 14d, the decolorization rate of the strain RG-171 inactivated fruiting body reaches the maximum value 45.83% at 14d, the COD removal rate reaches the maximum value 29.79% at 10d, the decolorization rate and the COD removal rate of the inactivated fruiting body respectively fluctuate between 20% -40% and 10% -30% in 14d, the decolorization rate of the non-inactivated fruiting body reaches the maximum value 63.78% at 10d, and the COD removal rate is obviously higher than that of the inactivated fruiting body after the 14d reaches the maximum value 75.04% and 7 d.
Example 4 degradation experiments with different wastewater addition amounts
The preparation method of the RG-171 strain liquid culture solution comprises the following steps:
activating RG-171 strain, taking bacterial blocks with the diameter of 0.5cm by using a puncher after hyphae grow on a flat plate, inoculating the bacterial blocks into a liquid culture medium, and carrying out shake culture at 25 ℃ for 7d at 150r/min to form bacterial balls with the same size for later use.
Based on the liquid culture solution of RG-171 strain, 1, 2, 3, 4 and 5mL of liquid are respectively removed from a conical flask in the liquid culture solution, the corresponding volume of wetland sewage is added, the total volume of the mixed solution is 20mL, the wastewater accounts for 5%, 10%, 15%, 20% and 25% of the total volume of the mixed solution, the mixed solution is subjected to shaking culture at 25 ℃ for 14d at 150r/min, and 3 parallel samples are arranged for each treatment. The day of adding the humidified sewage is day 0, and samples are taken at the 3d, 7d, 10d and 14d respectively for detection. COD value and chromaticity were measured, and the average value and standard deviation of 3 replicates were taken as final results. The strain RG-171 has the highest decoloring rate reaching 74.43 percent when 4mL of sewage is added for 14 d; when 4mL of sewage is added for 14d, the COD removal rate is highest and reaches 74.00%.
Example 5 laccase Activity Change experiment during degradation
RG-171 strain cultured in liquid medium was based (preparation method was same as in example 4), the day of adding wet ground sewage was day 0, shaking culture was conducted for 14d, and crude enzyme solutions were sampled and prepared for detection at the times of 2d, 4d, 6d, 8d, 10d and 14d, respectively. And (3) measuring laccase activity, COD value and chromaticity, centrifuging the final mycelium and fermentation broth at 4 ℃ and 12000rpm for 30min, filtering to obtain supernatant for analysis of subsequent degradation products, measuring biomass of the residual mycelium, and taking the average value and standard deviation of 3 parallel samples as a final result.
The change of laccase activity and decoloration rate and COD removal rate in the degradation process is shown in figure 2. As shown, the enzyme activity gradually increased over time during the growth of the strain. The extracted laccase activity was determined using a kit (Suzhou Ming Biotechnology Co., ltd.). Definition of laccase viability unit (U): the amount of enzyme required to oxidize 1. Mu. Mol of ABTS substrate per minute at a wavelength of 420 nm.
Strain RG-171 reached an enzyme peak at day 10, the peak of enzyme activity was 64.32U/L, after which the enzyme activity tended to stabilize or slightly decrease. The decolorization rate and COD removal rate reached a higher level on day 7 or 10d, and then tended to stabilize or slightly rise.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. Lung-shaped Pleurotus ostreatusPleurotus pulmonarius) RG-171, wherein said Pleurotus ostreatus RG-171 was deposited at the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12/08 of 2022; deposit unit address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation number: CGMCC No.40366.
2. A microbial agent comprising Pleurotus ostreatus RG-171 as defined in claim 1.
3. Use of the Pleurotus ostreatus RG-171 as defined in claim 1 or the microbial agent as defined in claim 2 in the treatment of wetland sewage.
4. Use according to claim 3, characterized in that the use is decolorization and COD degradation.
5. A method for treating wetland sewage, which is characterized by comprising the steps of inoculating the pleurotus pulmonarius RG-171 described in claim 1 or the microbial agent described in claim 2 into the wetland sewage for decolorization and COD degradation.
6. The method of claim 5, wherein the time for decolorizing and COD degradation is 14d.
7. A wetland sewage treatment agent, characterized by comprising the Pleurotus pulmonarius RG-171 according to claim 1 or the microbial agent according to claim 2.
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| CN108529760A (en) * | 2018-04-26 | 2018-09-14 | 东北农业大学 | The repairing method of microorganism that degradable organic wastewater is coupled with heavy metal polluted waste water |
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| CN108529760A (en) * | 2018-04-26 | 2018-09-14 | 东北农业大学 | The repairing method of microorganism that degradable organic wastewater is coupled with heavy metal polluted waste water |
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| Fungal treatment of humic-rich industrial wastewater: application of white rot fungi in remediation of food-processing wastewater;Mostafa Zahmatkesh 等;《ENVIRONMENTAL TECHNOLOGY》;第2752-2762页 * |
| 产漆酶杏鲍菇原生质体紫外线诱变育种及其发酵条件研究;刘敏;《万方》;全文 * |
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