Lactobacillus pentosus strain JN-R2 and application thereof in purifying sheep washing wastewater

Abstract

The application provides a lactobacillus pentosus JN-R2 strain, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, has a preservation number of CGMCC No. 25651 and a preservation date of September 5, 2022. The strain has a remarkable effect in the application of sheep wool washing wastewater treatment, can quickly demulsify and flocculate and precipitate emulsified lanolin, has a COD removal rate as high as 83.6%, and can efficiently remove EDCs pollutants to pollute the lanolin of sheep wool, so that the extracted lanolin has higher application safety. The lactobacillus pentosus JN-R2 strain provided by the application has lower lipase and oxidase activities, well retains the activity of lanolin, solves the defects and deficiencies in the prior art, and provides better technical support for efficiently treating wool washing wastewater and extracting lanolin.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of microbial technology and water treatment technology, in particular to a lactobacillus pentosus JN-R2 and its application in purifying sheep wool washing wastewater. BACKGROUND

[0002] Sheep wool washing wastewater is high-concentration organic wastewater discharged from wool washing production process, and is one of the most difficult pollution sources to treat in the world. The main components of the wastewater are lanolin, sheep sweat, sheep manure, soil, grass and sundries. The lanolin in the wastewater is in an emulsified state, and is covered with a layer of fat dregs containing various organic matters, fine suspended solids and various soluble organic matters, which are the main components of COD in the wastewater. The wastewater is difficult to treat, has high cost and is difficult to biodegrade, and is a problem that needs to be solved urgently in the textile industry.

[0003] Lanolin is a fat formed by a higher molecular weight fatty acid and approximately equal amounts of fatty alcohol, sterol, trimethyl sterol and the like, and contains a small amount of free acid, free alcohol, alkane and unknown substances. A small amount of crude lanolin is refined and directly used in the fields of medicine and cosmetics, rust inhibitor and the like. Most of the derivatives are made of wool alcohol and wool acid obtained by saponification of crude wool soap, and are widely used as additives in the medicine and cosmetic industries. The conventional method for extracting lanolin from wool washing wastewater is centrifugal separation, ultrafiltration membrane method, chemical / salt flocculation-extraction and the like. These methods not only have low efficiency, high input cost, cause secondary pollution and poor quality of the extracted lanolin, but also often ignore the fact that the prepared and extracted lanolin is mixed with a small amount of EDCs endocrine disruptors, which can be easily absorbed through the human skin and mouth, and further limits the wide application of lanolin in medicine and cosmetics.

[0004] As a new pollutant, endocrine disruptors (EDCs) have strong liposolubility, hydrophobicity, low-dose effect and long half-life, and the content of environmental estrogen (estriol, 17β-estradiol, bisphenol A, ethinyl estradiol and other estrogens) at the level of ng / L can cause the increase of infertility rate and incidence of reproductive system diseases, interfere with the endocrine system of the human body, cause infantile precocious puberty and threaten the health of the population. The livestock manure in the breeding industry is one of the important sources of environmental estrogen. At present, no single technology can effectively treat wool washing wastewater. Therefore, it is of great significance to find a technology that can not only effectively treat wool washing wastewater but also effectively remove EDCs pollution of lanolin to solve the problem of purifying wool washing wastewater and recycling lanolin.

[0005] TECHNICAL SCHEME

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] The application discloses a lactobacillus pentosus JN-R2, and relates to screening and identification of the strain, application of the lactobacillus pentosus JN-R2 in purifying sheep wool washing wastewater, and comparative experiments of different lactobacillus in application to the sheep wool washing wastewater.

[0008] Preferably, the lactobacillus pentosus JN-R2 is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 25651 and a preservation date of September 5, 2022.

[0009] Preferably, the screening of the strain comprises sampling, preliminary screening and rescreening.

[0010] Preferably, the identification of the strain comprises physiological and biochemical identification of the strain JN-R2, molecular biological identification of the strain JN-R2 and screening of the strain.

[0011] Preferably, the lactobacillus pentosus JN-R2 microbial preparation reduces the COD of the wool washing wastewater and removes EDCs pollutants dissolved in wool grease.

[0012] Preferably, the comparative experiments of different lactobacillus in application to the sheep wool washing wastewater comprise pretreatment of the wool washing wastewater and comparative experiments of different lactobacillus.

[0013] Compared with the prior art, the application provides the lactobacillus pentosus JN-R2 and application thereof in purifying sheep wool washing wastewater, and has the following beneficial effects:

[0014] The lactobacillus pentosus JN-R2 strain fermentation culture solution provided by the application has a remarkable treatment effect in the sheep wool washing wastewater, can quickly demulsify and separate the emulsified wool grease, simultaneously efficiently flocculates and precipitates the hydrophobic grease after demulsification, the wool washing wastewater grease precipitation rate reaches 85.5%, the COD removal rate of the wool washing wastewater reaches 83.6%, and the EDCs pollutants are efficiently removed from the wool grease, so that the application safety of the extracted wool grease is higher. The lactobacillus pentosus JN-R2 strain provided by the application has lower lipase and oxidase activities, and the activity of the wool grease is well reserved, so that the defects and deficiencies in the prior art are solved, and better technical support is provided for efficient treatment of wool washing wastewater and wool grease extraction. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A is a colony morphology diagram of the lactobacillus pentosus JN-R2, and 1B is a cell morphology diagram of the lactobacillus pentosus JN-R2 under a microscope.

[0016] Figure 2 The phylogenetic tree is constructed based on 16S rDNA sequences of the separated strain and other related strains collected in Genbank, and can be used for preliminary identification of the taxonomic position.

[0017] Figure 3 Figure 1 is a comparison result diagram of the flocculation of the Lactobacillus pentosus JN-R2 strain in a small test experiment of the present application. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0019] Please refer to Figures 1-3 The Lactobacillus pentosus JN-R2 includes the screening of the strain, the identification of the strain, the application of the Lactobacillus pentosus JN-R2 in purifying scouring wastewater, and the comparative experiment of different Lactobacillus applied in scouring wastewater.

[0020] Further, the screening of the strain includes sampling, preliminary screening, and rescreening.

[0021] Further, the identification of the strain includes physiological and biochemical identification of the strain JN-R2, and molecular biology identification of the strain JN-R2.

[0022] Further, the Lactobacillus pentosus JN-R2 fermentation liquid reduces the COD of scouring wastewater and removes EDCs pollutants dissolved in wool grease.

[0023] Further, the comparative experiment of different Lactobacillus applied in scouring wastewater includes pretreatment of scouring wastewater and comparative experiment of different Lactobacillus species.

[0024] The following will be further described through embodiments:

[0025] Embodiment 1: Screening of the strain

[0026] S1: Sampling: 10g of scouring wastewater sludge collected from an industrial park in Xingtai, Hebei Province was dispersed in 100ml of sterile normal saline, and cultured at 30℃ and 200rpm for 2h on a shaking table, as the original mixed bacterial liquid for standby. After standing, the supernatant was diluted by 10 times gradient, 200ul of bacterial suspension of different dilution was taken and respectively coated on the enrichment solid culture medium, and then cultured in a 30℃ incubator for 2d. Then single colonies were picked for further isolation and purification.

[0027] S2: Preliminary screening: Different single colonies after purification in step S1 were inoculated in enrichment liquid medium, first cultured at 30°C, 150 rpm for 1 day, then static cultured for 2 days, 10 ml of the culture fermentation broth was added into 100 ml of kaolin suspension with 4 g / L, at the same time, kaolin suspension without culture broth was used as control, and the flocculation time and degree were observed for 1 day, and the flocculation activity was determined by the flocculation degree, and six strains with high flocculation degree were obtained.

[0028] S3: Rescreening: The six strains with high flocculation degree in step S1 were coded as No. 1 strain, No. 2 strain, No. 3 strain, No. 4 strain, No. 5 strain and No. 6 strain, respectively, and 10 ml of the culture fermentation broth (cultured in enrichment liquid medium) was added into 100 ml of soybean oil wastewater containing 20 ug / L estradiol and sterilized in advance, and then stirred by a magnetic stirrer at 300 rpm for 20 min, and then placed in a sterile operation table for 24 h. Then the supernatant and flocculation product were separated by a low-speed centrifuge (5000 rpm). The removal rates of oil flocculation, COD and estradiol were detected after centrifugation, and the results are shown in Table 1. Since estradiol is easily dissolved in oil, it will be removed together with the oil during the oil removal process. Through the detection results, a strain No. 2 strain with high efficiency of oil demulsification, flocculation and sedimentation and little change in estradiol concentration in the supernatant after centrifugation was finally screened, which was named as JN-R2, and the strain was purified and frozen for preservation.

[0029] Table 1: Removal rates of various indicators of soybean oil wastewater after centrifugation

[0030] Detection item No. 1 bacteria No. 2 bacteria No. 3 bacteria No. 4 bacteria No. 5 bacteria No. 6 bacteria Oil removal rate % 23 85 52 35 12 42 COD removal rate % 21 81 43 25 8 35 Estrogen removal rate % 18 2 45 30 9 36

[0031] The components and proportions of the above-mentioned enrichment liquid medium are as follows: glucose 10 g / L, tryptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 5 g / L, ammonium sulfate 2 g / L, sodium chloride 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, sterilized at 121°C for 20 min and ready for use. The enrichment solid medium is the above-mentioned liquid medium added with 2 g / L agar.

[0032] The above-mentioned oil removal rate detection method adopts weighing method (GBT8538-2008); the COD detection method is detected by conventional digester-COD detector; the estradiol detection method adopts conventional solid phase extraction (SPE)-high performance liquid chromatograph (HPLC) / fluorescence detector (FLD) detection;

[0033] Example 2: Identification of the strain

[0034] The strain JN-R2, obtained through the above isolation and purification, exhibits highly efficient demulsification and flocculation effects while maintaining a constant estriol concentration. After culturing this strain on enriched solid medium at 30℃ for 48 hours, its colonies are raised, smooth, milky white, round, with neat edges, and a diameter of approximately 1 mm. Figure 1 A) The cell morphology of the strain under the microscope is as follows: Figure 1 As shown in Figure B, the bacteria are Gram-positive but lack spores and flagella.

[0035] S1: Physiological and biochemical identification of strain JN-R2 (Table 2)

[0036] Table 2: Physiological and Biochemical Identification Results

[0037] Biochemical index Results Biochemical index Results Lipase - Glucose + Catalase - Sucrose + Contact enzyme - Galactose + Oxidase - Lactose + Gelatin liquefaction - Xylose + V.P test + Arabinose + 6.5% salt tolerance + Cellobiose +

[0038] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0039] S2: Molecular biological identification of strain JN-R2 was performed by amplifying 16S rDNA using universal bacterial primers.

[0040] Primer: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0041] 1492R:5'-TACGACTTAACCCCAATCGC-3'

[0042] Reaction system: 12.5 μL of 2×TaqPCR Master Mix, 1 μL each of forward and reverse primers, 0.5 μL of DNA template, and 10 μL of ddH2O; Reaction program: 95℃ for 5 min; 94℃ for 1 min, 55-58℃ for 1 min, 72℃ for 90 s, 30 cycles; 72℃ for 10 min.

[0043] S3: The PCR product obtained in step S1 was analyzed by agarose gel electrophoresis and then sent to Shanghai Sangon Biotech for sequencing. Its 16S sequence is shown in SEQ ID NO.1. BLAST sequence alignment was performed on the NCBI website, and the results showed 99% homology with the registered *Lactobacillus pentosus* sequence, confirming the strain as *Lactobacillus pentosus*. The selected strain was named *Lactobacillus pentosus* JN-R2. Figure 2 A phylogenetic tree was constructed based on the 16S rDNA sequences of the isolated strain and other related strains included in Genbank.

[0044] Lactobacillus pentosus JN-R2, which was preserved in China General Microbiological Culture Collection Center (CGMCC) on September 5, 2022, and the address of the CGMCC is No. 1, Beichen West Road, Haidian District, Beijing, China, and the preservation number is CGMCC No. 25651.

[0045] Example Three: Application of Lactobacillus pentosus JN-R2 in purifying scouring wool wastewater

[0046] S1: The pretreatment method of the scouring wool wastewater was to mix the collected scouring wool wastewater from an industrial park in Xingtai, Hebei Province uniformly, stand for 24 h, take the upper liquid to be sterilized at 121℃ for 20 min, and then use it as the scouring wool wastewater to be treated in the laboratory. The total concentration of EDCs in the scouring wool wastewater was 15.3 ng / L, including 6 ng / L of estratriol, 5.7 ng / L of 17β-estradiol, 2 ng / L of bisphenol A, and 1.6 ng / L of ethinyl estradiol, which was detected by solid phase extraction (SPE)-high performance liquid chromatograph (HPLC) / fluorescence detector (FLD).

[0047] S2: Take the separated and purified Lactobacillus pentosus JN-R2 strain, inoculate it in 100 ml of fresh lactic acid liquid medium, and ferment it at 30℃ under static conditions for 2 d. Take 10 ml of the fermentation culture liquid and add it to 100 ml of the scouring wool wastewater, and the PH of the mixture is 5.0. At the same time, first adjust the PH of the scouring wool wastewater to 5.0 by using 10% sulfuric acid to break the emulsion, and then take 10 ml of polyaluminum sulfate flocculant with a concentration of 0.4 g / L and add it to the 100 ml of scouring wool wastewater after emulsion breaking as a control experiment. The experimental group and the control group are both stirred at 300 rpm by a magnetic stirrer for 20 min, and then placed in a sterile operation table for 48 h. The supernatant and flocculation product of the scouring wool wastewater are separated by using a low-speed centrifuge (5000 rpm), and the oil flocculation precipitation removal rate, COD removal rate, and EDCs concentration of the scouring wool wastewater after centrifugation, as well as the EDCs concentration of the flocculation product are detected, and the results are shown in Table Three.

[0048] Table Three: Results of treating scouring wool wastewater with Lactobacillus pentosus JN-R2 strain culture liquid

[0049]

[0050] As shown by the experimental results, the Lactobacillus pentosus JN-R2 culture solution provided by the application can produce demulsification and flocculation and precipitation of oil in wool scouring wastewater, the precipitation effect is lower than that of the polyaluminum sulfate flocculant, but the COD removal rate of the wool scouring wastewater is as high as 85.5%. The experiment also shows that the EDCs pollutant concentration of the supernatant after saponification is obviously higher than that of the supernatant without saponification, and the EDCs pollutant concentration in the flocculated oil product is almost zero. It is indicated that the EDCs pollutants in the supernatant have esterification reaction with certain acidic substances in the Lactobacillus pentosus JN-R2 fermentation solution, the lipid compound has the characteristics of being extremely soluble in water, and in the flocculation and precipitation process, the lipid compound which is extremely soluble in water is effectively separated from the oil in the wool scouring wastewater, the influence of the removal of EDCs on the wool grease pollution is realized, and the safety application of the subsequent extraction of crude lanolin is ensured.

[0051] The above lactic acid liquid culture medium: 10 g / L of proteose peptone, 5 g / L of beef extract powder, 4 g / L of yeast extract powder, 20 g / L of glucose, 1.0 ml / L of Tween-80, 2 g / L of potassium phosphate dibasic, 5 g / L of sodium acetate, 2.0 g / L of triammonium citrate, 0.2 g / L of magnesium sulfate (MgSO4.7H2O), 0.2 g / L of manganese sulfate (MnSO4.4H2O), final PH 6.2±0.2, sterilized at 121℃ for 20 min, and then used.

[0052] The above supernatant saponification treatment process: 50 ml of the supernatant is concentrated by 10 times in a water bath, 10 ml of anhydrous ethanol is added, 3 g of sodium hydroxide solid is added, and the mixture is placed in a round-bottom flask and heated by alcohol through a asbestos screen for 10 min to obtain a mixed solution, and the EDCs pollutants are detected.

[0053] The above flocculation product saponification treatment process: all the flocculation precipitates are taken, 5 ml of deionized water and 10 ml of anhydrous ethanol are added, 3 g of sodium hydroxide solid is added, and the mixture is placed in a round-bottom flask and heated by alcohol through a asbestos screen for 10 min to obtain a mixed solution, and the EDCs pollutants are detected.

[0054] Example Four: Comparative experiment of different Lactobacillus applied to wool scouring wastewater

[0055] S1: The pretreatment method of the wool scouring wastewater is that the wool scouring wastewater collected from an industrial park in Qinghe County, Xingtai, Hebei is stirred uniformly, and the upper liquid is taken after standing for 24 h and sterilized at 121℃ for 20 min to serve as the wool scouring wastewater to be treated in the laboratory. The total EDCs concentration of the wool scouring wastewater is 15.3 ng / L, including 6 ng / L of estratriol, 5.7 ng / L of 17β-estradiol, 2 ng / L of bisphenol A and 1.6 ng / L of ethinyl estradiol, which is detected by solid phase extraction (SPE)-high performance liquid chromatograph (HPLC) / fluorescence detector (FLD).

[0056] S2: Take the separated and purified Lactobacillus pentosus JN-R2 strain, inoculate in 100 ml of fresh lactic acid liquid medium, and carry out fermentation under static condition at 30 DEG C for 2 d, and culture Lactobacillus pentosus (number BNCC337069), Lactobacillus plantarum (number BNCC134428), Lactobacillus acidophilus (number BNCC134426), Lactobacillus rhamnosus (number BNCC134266) and Lactobacillus casei (number BNCC134415) under the same medium and fermentation condition, which are all purchased from China Industrial Microbial Culture Collection Center (CICC) and are control experiments. Take 10 ml of the fermentation liquor of each strain prepared above, add to 100 ml of scouring wastewater, and stir for 20 min by a magnetic stirrer at 300 rpm, and then place in a sterile operation table for 48 h. The supernatant and flocculation product of the scouring wastewater are separated by a low-speed centrifuge (5000 rpm), and the removal rate of grease flocculation and precipitation, the removal rate of COD and the concentration of EDCs of the scouring wastewater after centrifugation, and the concentration of EDCs of the flocculation product are detected, and the results are shown in Table 4.

[0057] Table 4: Comparison results of different lactic acid bacterial strains

[0058]

[0059] As seen from the purification treatment results of the scouring wastewater by different lactic acid bacterial strains, the grease settlement rate and the COD removal rate of each lactic acid bacteria in the control group are lower than those in the experimental group, and the EDCs concentration after saponification of the flocculation product is higher than that in the experimental group, which indicates that the lactic acid bacteria JN-R2 provided in the application has higher grease demulsification and precipitation performance and higher COD removal rate performance for the grease in the scouring wastewater, and the EDCs pollutants have the lowest pollution to the wool grease. The comparison chart of the flocculation small test of the control group Lactobacillus pentosus (number BNCC337069) and the experimental group Lactobacillus pentosus JN-R2 is shown in FIG. 1. Figure 3

[0060] As seen from the comparison and analysis of the grease precipitation rate and the EDCs concentration, since the fermentation liquor of each lactic acid bacteria in the control group has various lipase or oxidative decomposition enzyme activities, the recovery rate of the flocculation and precipitation grease in the control group is low, and the EDCs pollutants also have the phenomenon of oxidative decomposition, while the fermentation culture liquor of the Lactobacillus pentosus JN-R2 provided in the application has lower degradation or oxidation to the grease in the scouring wastewater, which ensures the high recovery rate of the extracted wool grease. The EDCs in the scouring wastewater treated by the application can be removed by adding other aerobic bacteria or oxidation preparations, and the treated wastewater can be repeatedly used for washing wool.

[0061] ​The above lactic acid liquid culture medium: proteose peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, Tween-80 1.0 ml / L, potassium phosphate dibasic 2 g / L, sodium acetate 5 g / L, triammonium citrate 2.0 g / L, magnesium sulfate (MgSO4.7H2O) 0.2 g / L, manganese sulfate (MnSO4.4H2O), final PH 6.2±0.2, sterilized at 121°C for 20 min and then stored.

[0062] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A strain of Lactobacillus pentosus JN-R2, characterized in that The lactobacillus pentosus JN-R2 strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 25651, and the preservation date is September 5, 2022.

2. The microbial preparation produced by the lactobacillus pentosus JN-R2 according to claim 1, wherein the active ingredient is the lactobacillus pentosus JN-R2.

3. The application of Lactobacillus pentosus JN-R2 in purifying the waste water of washing sheep wool according to claim 1, characterized in that The wool washing wastewater is high concentration organic wastewater produced in the wool washing process, and the COD value is between 4x10 4 10X10 4 mg / L.

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