CN110438033B

CN110438033B - Grease degrading bacterium, application and grease degrading method

Info

Publication number: CN110438033B
Application number: CN201910590315.9A
Authority: CN
Inventors: 柳志强; 王艳梅; 周海岩; 薛亚平; 柯霞; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-07-02
Filing date: 2019-07-02
Publication date: 2021-04-27
Anticipated expiration: 2039-07-02
Also published as: CN110438033A

Abstract

The invention discloses a grease degrading bacterium, application and a grease degrading method. The oil degrading bacteria are classified and named as Brevundimonas vannanneytii DK2, and the preservation number is CCTCC NO: M2019332. The invention separates a strain capable of efficiently degrading oil from humus, the strain is classified and named as Brevundimonas vannanneytii DK2, the preservation number is CCTCC NO: M2019332, the strain has strong environmental adaptability and simple nutritional requirement, can survive and degrade oil which is an important component in kitchen waste compost, and under the prior art, the degradation rate of edible blend oil can reach 60 percent, and the degradation rate of crude fat in kitchen waste reaches more than 45 percent. Has better application prospect.

Description

Grease degrading bacterium, application and grease degrading method

Technical Field

The invention relates to the technical field of kitchen waste treatment, in particular to an oil degrading bacterium, application and an oil degrading method.

Background

With the rapid increase rate of daily output of the kitchen waste in China, the traditional incineration and landfill technology in China cannot solve the problems of low degradation efficiency, large floor area, serious environmental pollution and the like of the kitchen waste, and the dilemma that China is in a 'waste enclosure' is caused. Therefore, the application of the composting technology to the treatment of the kitchen waste is urgently needed. However, due to the characteristic that the main component of the kitchen waste is difficult to degrade, the degradation of the grease becomes a key speed-limiting step for the composting application of the kitchen waste, so that the introduction of a means of specifically degrading the grease by using an exogenous bacterial source is particularly important in the rapid degradation of the kitchen waste.

Cinchoping et al (CN102586112A) degrade kitchen waste by using Bacillus and Pseudomonas as fat degrading bacteria and starch degrading bacteria, cellulose degrading bacteria and protein degrading bacteria as microbial agents, and the waste degradation rate is 78% after 10 days; song Jian et al (CN 103756941A) take Bacillus licheniformis as a fat degrading bacterium to act with other degrading bacteria synergistically to obtain the kitchen waste with the reduction rate of 91%; li, T, etc. (Waste Manag 34(12):2641-6) inoculating bacillus and pseudomonas into kitchen Waste, and when composting is completed for 42d, the Waste degradation rate reaches 85-90%; in terms of the compost reduction of the kitchen waste, the inoculation of the microorganisms can provide a good idea for the treatment of the kitchen waste in the city enclosed by China, but the defect of long time consumption also becomes a key problem. In the composting process, whether the degradation rate of the grease is improved by the external microorganisms or not is not specifically researched, so that the speed-limiting step in the degradation process of the kitchen waste is overcome.

On the other hand, Joo (Environ Pollut,156(3):891-6) introduces Candida catenulata to treat the mixture of the kitchen waste and the soil polluted by crude oil, and after 13 days of composting, the degradation rate of petroleum hydrocarbon substances is found to reach 84 percent, which is improved by 36 percent compared with the control group without inoculation; the crude fat is reduced by 30.7 percent and is improved by 29.3 percent compared with a control group which is not inoculated after 4 kinds of bacillus are prepared into microbial inoculum and thrown into the kitchen waste for 24 hours by Lei Hua Zhi and the like (university of east China (Nature science edition), 2011(2): 126-. The work of screening microbial resources to degrade grease and restore land polluted by crude oil is reported at home and abroad, but the application of the microbes to kitchen waste treatment and real application to life is less.

Therefore, in order to efficiently degrade the grease, remove the limit of the kitchen waste in the degradation process, accelerate the composting process of the kitchen waste, improve the fermentation efficiency, shorten the fermentation time, reduce the treatment cost, and screen the strains which can adapt to different environments and have good synergistic effect with other strains, the method has important value and practical significance.

Disclosure of Invention

The invention provides a novel grease degrading bacterium and application of the grease degrading bacterium in grease degradation.

The invention obtains a new oil degradation bacterium by screening, which is obtained by enriching once from humus, acclimating for seven rounds in an inorganic salt culture medium containing a unique carbon source, namely edible oil, primarily screening by a screening culture medium flat plate, and then transferring to an oil-containing culture medium for secondary screening. Classified and named as Vancaneytii Brevundimonas (Brevundimonas vannanneytii) DK2 (short for DK2), and is preserved in China center for type culture Collection with the preservation number: CCTCC NO, M2019332, preservation date: year 2019, 5 month, 7 days, deposit address: wuhan university in Wuhan, China.

The Brevundimonas vannanneytii DK2 capable of efficiently degrading the grease, which is obtained by screening, is cultured on an LB solid plate for 14h, the bacterial colony is grayish white, slightly transparent, and smooth and mellow in surface, and the bacterial colony appears grayish yellow after 36 h. Under an electron microscope, the cell is rod-shaped, has no spores and has flagella. Through molecular biological identification and Biolog microbial identification results, the strain is considered to belong to Brevundimonas vannanneytii.

The invention also provides application of the grease degrading bacteria in grease degradation. The oil is oil in the kitchen waste or edible blend oil.

The invention also provides a method for degrading the grease, which comprises the following steps: and (3) taking oil as a carbon source, adding an inorganic salt basic culture medium as a fermentation culture medium, inoculating the oil degrading bacteria, and then carrying out fermentation culture.

Preferably, the conditions of the fermentation culture are: the temperature is 30-65 ℃, and the rotating speed is 150 r/min. The most suitable temperature is 50 ℃.

Preferably, the addition amount of the grease degrading bacteria is 20-100 g of wet bacteria per liter of inorganic salt basic culture medium. The most suitable amount is 40 g.

More preferably, the preparation method of the wet bacteria of the oil-and-fat degrading bacteria comprises the following steps: inoculating the oil degrading bacteria into an LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12-14h to obtain a seed solution; transferring the seed solution into a 500mL triangular shake flask filled with 100mL LB liquid culture medium by an inoculation amount with a volume concentration of 1%, culturing at 37 ℃ and 150rpm for 12-14h to obtain a fermentation liquid, centrifuging the fermentation liquid at 4 ℃ and 8000rpm for 10min, removing supernatant, and collecting wet thalli.

Preferably, the addition amount of the grease is 20-100 g per liter of the inorganic salt basic culture medium. The most suitable amount is 40 g.

Preferably, the inorganic salt basal medium comprises the following components: 1.5g/L of ammonium sulfate, 2g/L of sodium chloride, 2g/L of disodium hydrogen phosphate dodecahydrate, 2g/L of potassium dihydrogen phosphate and 0.5g/L of magnesium sulfate heptahydrate, wherein the solvent is deionized water and the pH value is 4-9. The pH is most suitably 5.5.

The invention separates a strain capable of efficiently degrading oil from humus, the strain is classified and named as Brevundimonas vannanneytii DK2, the preservation number is CCTCC NO: M2019332, the strain has strong environmental adaptability and simple nutritional requirement, can survive and degrade oil which is an important component in kitchen waste compost, and under the prior art, the degradation rate of edible blend oil can reach 60 percent, and the degradation rate of crude fat in kitchen waste reaches more than 45 percent. Has better application prospect.

Drawings

FIG. 1 shows the morphology of a colony cultured for 16h (A) and the morphology of the colony cultured for 48h (B) with the strain DK 2.

FIG. 2 is an electron micrograph of strain DK2, wherein A is an SEM electron micrograph (1200K X) and B is a TEM electron micrograph (20000X).

FIG. 3 is a phylogenetic tree result of strain DK 2.

Fig. 4 is a graph of the detection result of the change of the degradation rate of the strain DK2 on grease under different conditions, wherein a: different temperatures, B: inoculation amount and C: substrate concentration, D: the pH value.

FIG. 5 shows the application of Brevundimonas vannanneytii DK2 in degrading crude fat in kitchen waste. (wherein CK was not additionally inoculated with a microorganism, representing a control group.)

Detailed Description

Culture medium:

the enrichment medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of edible blend oil and 6g/L of Tween 80, wherein the solvent is deionized water, and the pH value is natural. Sterilizing at 115 deg.C for 30 min.

The domestication culture medium comprises: 1g/L dipotassium phosphate, 1g/L potassium dihydrogen phosphate, 1g/L disodium phosphate, 0.5g/L sodium chloride, 0.5g/L magnesium sulfate heptahydrate, deionized water as a solvent and natural pH value. Sterilizing at 121 deg.C for 20 min.

The oil screening culture medium comprises the following components: 2g/L of ammonium sulfate, 1g/L of dipotassium phosphate, 0.5g/L of potassium chloride, 0.5g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate, 20g/L of agar, 12mL/L of edible blend oil emulsion, deionized water as a solvent and natural pH. Sterilizing at 121 deg.C for 20 min. Wherein the edible blend oil emulsion is obtained by emulsifying edible blend oil and 20g/L polyvinyl alcohol water solution at a volume ratio of 1:3 with a high-pressure homogenizer (pressure of 0.4MPa) twice.

The inorganic salt culture medium comprises the following components: 1.5g/L of ammonium sulfate, 2g/L of sodium chloride, 2g/L of disodium hydrogen phosphate dodecahydrate, 2g/L of potassium dihydrogen phosphate and 0.5g/L of magnesium sulfate heptahydrate, wherein the solvent is deionized water, and the pH value is natural. Sterilizing at 121 deg.C for 20 min.

Slant medium/purification medium (YPD medium) composition: 10g/L of yeast powder, 20g/L of peptone, 20g/L of glucose and 20g/L of agar, wherein the solvent is deionized water, and the pH value is natural. Sterilizing at 115 deg.C for 30 min.

LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride, wherein the solvent is deionized water, and the pH value is natural. Sterilizing at 121 deg.C for 20 min.

Example 1

Sampling and enriching: the sample was taken from humus soil near the canteen of university of Zhejiang industry, 5g of each soil sample (wet weight) was weighed and inoculated into a 250mL triangular flask containing 50mL of enrichment medium, and cultured on a shaker at 30 ℃ and 150rpm for 72 h.

Domestication: filtering the enriched sample with three layers of gauze, putting 25mL of bacterial liquid into an acclimatization culture medium containing 5g/L edible blend oil, and culturing for 4 days on a shaking table at 37 ℃ and 150 rpm; taking 25mL of culture solution domesticated in domestication culture medium of 5g/L edible blend oil for 4 days, and culturing in domestication culture medium containing 10g/L edible blend oil at 37 deg.C and 150rpm on a shaking table for 4 days; taking 4d as a period, after domestication of each period is completed, taking 25mL of culture solution into a domestication culture medium, and adding 5g/L of oil content in each round until domestication in a culture medium containing 25g/L of edible blend oil is completed.

Dilution coating: 1mL of domesticated bacterial liquid is taken to be diluted in 9mL of normal saline in a gradient way, and the dilution is respectively taken to be 10⁴-10⁷The diluted solution (100 mu L) is coated on a grease screening culture medium plate and cultured in a constant temperature incubator at 28 ℃ for 3-4 d.

And (4) purification and preservation: and (4) picking a single colony with an obvious transparent ring around the grease screening plate by using an inoculating loop, and streaking and purifying on a purification culture medium until the cell morphology under a microscope is consistent. And (3) selecting the purified single colony on a slant culture medium, culturing for 1-2 days at 37 ℃, and preserving in a refrigerator at 4 ℃ for later use.

And (3) determining the degradation rate: respectively taking a ring of the strains preserved on the inclined plane, inoculating the strains into a test tube filled with 5mL of LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12-14h to obtain seed liquid; transferring 1mL of the seed solution into a 500mL triangular shake flask filled with 100mL of LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12-14h to obtain a fermentation liquid. The fermentation broth was centrifuged at 8000rpm for 10min at 4 ℃ to obtain wet cells. The wet cells were inoculated at 20g/L into a 250mL Erlenmeyer flask containing 50mL of an inorganic salt medium containing 40g/L of an edible blend oil, and reacted at 37 ℃ and 150rpm for 7 d. After the reaction is finished, extracting the reaction solution twice by using 50mL of n-heptane, collecting the organic solvent-oil layer in a beaker with constant weight, drying the n-heptane in an oven at 75 ℃ until the weight is constant, weighing the weight by using an analytical balance, and calculating the degradation rate. Finally, a re-screening strain DK2 with higher degradation rate is obtained for slant preservation.

Example 2

Identification of strain DK 2.

1. And (3) observing colony morphology:

the slant-deposited strain of example 1 was picked in a small amount using an inoculating loop, streaked on a YPD plate, and cultured at 37 ℃ for 1 to 2 days, and the colony morphology is shown in FIG. 1. The bacterial colony is gray white, slightly transparent, smooth and mellow in surface, and gray yellow after 36 h.

2. And (3) SEM scanning electron microscope and TEM transmission electron microscope observation:

the cell morphology was rod-shaped, acapsular, spore (fig. 2, a), flagellar (fig. 2, B).

3. And (3) molecular identification:

preparing a template: single colonies were picked in 20. mu.L sterile ddH₂In an EP tube of O, boiled in water for 10min, centrifuged at 12000rpm for 1min, and the supernatant was used as a DNA template. 16S rDNA sequences were PCR amplified using 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-AAGGAGGTGATCCAGCCGCA-3') as forward and reverse primers.

The PCR amplification system was (50. mu.L): primers 27F and 1492R each 1. mu.L, DNA template 1. mu.L, ddH₂O22. mu.L, Master Mix enzyme 25. mu.L.

And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 40s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min, 35 cycles, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.

The sequence (SEQ ID NO.1) obtained was searched on the NCBI website by BLAST, rDNA sequences having high similarity to the sequence were selected, and the neighbor joining tree (NJ phylogenetic tree) was generated in 1000 replications by using MEGA7 software Align by Clustalw automatic analysis (FIG. 3), and the results showed that the strain DK2 had the highest homology to Brevundimonas (Brevundimonas banus Bannenris FDRGB2b) (homology, 97%/bp, based on 16S rDNA), Brevundimonas (Brevundimonas vannenytii LMG233 2337T) (homology, 98%/bp, based on 16S rDNA).

4. Biolog physiological and biochemical identification of microorganisms

Strains were phenotyped 94 strains using the Biolog (GEN iii) automated microorganism identification system, including 71 carbon source utilization assays and 23 chemosensitivity assays: inoculating strain DK2 into BUG plate culture medium (BIOLOG UNIVERSAL GROWTH AGAR), culturing at 33 deg.C for 2 days, washing thallus on the plate with sterile cotton swab, mixing with inoculating liquid (IF-A), making into bacterial suspension, and adjusting to 91% T/IF-A with turbidimeter. The bacterial suspensions were added to each well of Biolog GEN iii microwell assay plates using an 8-well electric applicator, 100 μ L per well. The plate was placed in a 33 ℃ incubator and read on a Biolog reader after 12h, 24h, 36h, 48h incubation, respectively.

The metabolic fingerprint is analyzed by a Biolog reading instrument, the strain DK2 can strongly utilize 17 carbon sources, and cannot utilize or has weaker utilization capacity on other 54 carbon sources; the strain is sensitive to 14 chemicals. The Biolog system gave an identification result of 36h and the similarity of the strain DK2 to Brevundimonas vannanneytii (BIOLOG system from model bacteria) was 0.808.

TABLE 1 ability of Strain DK2 to utilize 71 carbon sources on Biolog GEN III plates

TABLE 2 chemosensitivity of Strain DK2 to 23 chemicals on Biolog GEN III plates

The strain DK2 is preliminarily identified as Brevundimonas vannanneytii by combining morphological identification, molecular biology and Biolog microbial physiological and biochemical identification. The strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2019332 and the preservation date of 2019, 5 months and 7 days, and is classified and named as Brevundimonas vannanneytii DK 2.

Example 3

Application of Brevundimonas vannanneytii DK2 in degrading edible blend oil.

The experimental method comprises the following steps:

selecting a loop of the strain DK2 preserved on the inclined plane, culturing in a test tube filled with 5mL of LB liquid culture medium at 37 ℃ and 200rpm for 12-14h to obtain seed liquid; transferring 1mL of the seed solution into a 500mL triangular shake flask filled with 100mL of LB liquid culture medium, and culturing at 37 ℃ and 150rpm for 12-14h to obtain fermentation liquor. The fermentation broth was centrifuged at 8000rpm for 10min at 4 ℃ and wet biomass was collected for further use.

Temperature optimization: respectively inoculating wet thalli into an inorganic salt culture medium containing 40g/L edible blend oil according to the inoculation amount of 20g/L, reacting for 7d at 30-65 ℃ (5 ℃ is a gradient), extracting reaction liquid twice by 50mL of n-heptane, collecting an organic solvent-oil layer in a beaker with constant weight, drying the n-heptane in an oven at 75 ℃ until the weight is constant, weighing the weight by an analytical balance, and calculating the degradation rate.

And (3) optimizing the inoculation amount: respectively inoculating wet bacteria according to the inoculation amounts of 20g/L, 40g/L, 60g/L, 80g/L and 100g/L into an inorganic salt culture medium (pH6.5) containing 40g/L edible blend oil, reacting for 7d at 37 ℃ and 150rpm, extracting the reaction solution twice by using 50mL of n-heptane, collecting an organic solvent-oil layer in a beaker with constant weight, drying the n-heptane in an oven at 75 ℃ until the weight is constant, weighing the weight by analysis, and calculating the degradation rate.

Optimizing the concentration of the substrate: adding edible blend oil into an inorganic salt culture medium (pH6.5) at the concentrations of 20g/L, 40g/L, 60g/L, 80g/L and 100g/L, respectively inoculating wet thalli into inorganic salt culture media with different substrate concentrations according to the inoculation amount of 20g/L, reacting at 37 ℃ and 150rpm for 7d, extracting a reaction solution twice by using 50mL of n-heptane, collecting an organic solvent-oil layer in a beaker with constant weight, evaporating the n-heptane in a 75 ℃ oven until the weight is constant, weighing the weight by using an analytical balance, and calculating the degradation rate.

Optimizing the pH: adjusting the pH value of an inorganic salt culture medium by using HCl and NaOH (4, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0), inoculating wet bacteria into the inorganic salt culture medium containing 40g/L edible blend oil according to the inoculation amount of 20g/L, reacting for 7d at 37 ℃ and 150rpm, extracting a reaction solution twice by using 50mL of n-heptane, collecting an organic solvent-oil layer into a beaker with constant weight, drying the n-heptane in a 75 ℃ oven until the weight is constant, weighing the weight by using an analytical balance, and calculating the degradation rate.

The degradation rate of the oil is (A-B)/A x 100%.

Wherein:

a: oil content before inoculation, g;

b: oil content after reaction, g.

The results of each optimization are shown in FIG. 4.

The optimal inoculation amount of the Brevundimonas vannanneytii DK2 in the degradation process of the edible blend oil is 40 g/L; the optimum substrate concentration was 40 g/L.

The pH value of the kitchen waste in China is changed between 5.0 and 6.0, and the composting temperature is 50 to 60 ℃ in the aerobic composting process of the kitchen waste. The results show that the Brevundimonas vannanneytii DK2 has good degradation efficiency at the pH value of 5.5-7.0, the optimum pH value is 5.5, and the acid resistance is high; and the Brevundimonas vannanneytii DK2 can reach more than 50% of oil degradation rate under the reaction condition of 40-55 ℃, and the optimal temperature is 50 ℃, which shows that Brevundimonas vannanneytii DK2 resists high temperature.

Example 4

Application of Brevundimonas vannanneytii DK2 in degrading crude fat in kitchen waste.

The experimental method comprises the following steps:

preparing wet thalli: the same as in example 3.

Pretreating kitchen waste: collecting the kitchen waste of Zhejiang industrial university Jinghong dining hall, and crushing by using a crusher (FSJ-N05A 6).

Experimental groups: 200g (wet weight) of homogenized kitchen waste is weighed into a 500mL shake flask, 5% (wet weight) of DK2 wet thalli is inoculated, and the reaction is carried out at 50 ℃ and 150 rpm.

Control group: 200g (wet weight) of homogenized kitchen waste is weighed into a 500mL shake flask and reacted at 50 ℃ and 150 rpm.

Crude fatThe fat content determination method comprises the following steps: weighing empty extraction cup mass (m)₁) Pouring 50ml of petroleum ether (the boiling point is 30-60 ℃) into the leaching cup, building a device (a fat analyzer SOX406), weighing a certain mass of sample (n), placing the sample (n) into a filter paper cylinder, immersing the filter paper cylinder in the petroleum ether, turning on condensed water, turning on a heater, setting the temperature at 40 ℃ for 200 min. After heating, screwing on the cock, volatilizing petroleum ether at 50 deg.C, taking out the extraction cup after 30min, oven at 60 deg.C for 20min, taking out the extraction cup and weighing mass (m)₂)。

Wherein the crude fat content (% wet weight) — (m)₂-m₁)/n×100

The samples of the control group and the blank group reacted for 1d-8d were weighed respectively for crude fat content determination, and the results are shown in FIG. 5.

The results show that the un-inoculated kitchen waste is naturally fermented, and the oil degradation capability is lower and is not higher than 20%; the kitchen waste inoculated with Brevundimonas vannanneytii DK2 reacts at the 8 th day, and the oil degradation rate reaches 50.3 percent.

Sequence listing

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Claims

1. A grease degrading bacterium DK2, characterized in that the classification of the strain DK2 is named as a paratype brevundimonas (Brevundimonas sp.) (Brevundimonas vancanneytii) The preservation number is CCTCC NO: M2019332.

2. The use of the oil-and-fat-degrading bacterium DK2 according to claim 1 for degrading oil and fat.

3. The use according to claim 2, wherein the grease is grease in kitchen waste.

4. The use according to claim 2, wherein the oil is an edible blend oil.

5. A method for degrading grease is characterized by comprising the following steps: the method comprises the steps of taking oil as a carbon source, adding an inorganic salt basic culture medium, using the oil as a fermentation culture medium, inoculating the oil-degrading bacteria DK2 as claimed in claim 1, and then carrying out fermentation culture.

6. The method according to claim 5, wherein the fermentation culture is carried out under conditions selected from the group consisting of: the temperature is 30-65 ℃, and the rotating speed is 150 r/min.

7. The method for degrading oil and fat according to claim 5, wherein 20 to 100g of wet cells per liter of the inorganic salt basal medium is added to the oil and fat-degrading bacteria DK 2.

8. The method for degrading oil and fat according to claim 7, wherein the wet cell mass of oil and fat-degrading bacteria DK2 is prepared by: inoculating the oil-and-fat-degrading bacteria DK2 of claim 1 into an LB liquid culture medium, culturing at 37 ℃ and 200rpm for 12-14h to obtain a seed solution; transferring the seed solution into a 500mL triangular shake flask filled with 100mL LB liquid culture medium by an inoculation amount with a volume concentration of 1%, culturing at 37 ℃ and 150rpm for 12-14h to obtain a fermentation liquid, centrifuging the fermentation liquid at 4 ℃ and 8000rpm for 10min, removing supernatant, and collecting wet thalli.

9. The method for degrading oil and fat according to claim 5, wherein the amount of the oil and fat added is 20 to 100g per liter of the inorganic salt basic medium.

10. The method of degrading fats and oils according to claim 5, wherein the composition of the inorganic salt basal medium is: 1.5g/L of ammonium sulfate, 2g/L of sodium chloride, 2g/L of disodium hydrogen phosphate dodecahydrate, 2g/L of potassium dihydrogen phosphate and 0.5g/L of magnesium sulfate heptahydrate, wherein the solvent is deionized water and the pH value is 4-9.