CN113249275B

CN113249275B - Klebsiella variicola and application thereof

Info

Publication number: CN113249275B
Application number: CN202110750328.5A
Authority: CN
Inventors: 王清
Original assignee: Individual
Current assignee: Shandong Longze Shengtong Environmental Engineering Co ltd
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2022-04-26
Anticipated expiration: 2041-07-02
Also published as: CN113249275A

Abstract

The invention relates to Klebsiella variicola (Klebsiella variicola) KV-8, which is preserved in China center for type culture Collection in 1 month and 7 days of 2021, with the preservation address as follows: the preservation number of the eight-path Lojia mountain Wuhan university in Wuchang district, Wuhan city, Hubei province is CCTCC NO: m2021023; the Klebsiella variicola KV-8 has the characteristics of salt resistance, alkali resistance, high chlorine resistance and the like, and the removal of COD by the activated sludge of the epoxypropane saponified sewage added with the Klebsiella variicola KV-8 has obvious improvement effect.

Description

Klebsiella variicola and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to Klebsiella variicola and application thereof.

Background

At present, the domestic treatment of the propylene oxide wastewater is mainly a biological method, and the wastewater is cooled, settled, aerated and the like to reach the national discharge standard of sewage. The most important link is to degrade organic pollutants in sewage by microorganisms in activated sludge, but in general, high-concentration chlorine-containing wastewater has toxic action on the microorganisms, which is mainly reflected in that the high osmotic pressure of the wastewater destroys cell membranes and intramembrane enzymes of the microorganisms, so that the metabolic capacity of strains of the microorganisms is influenced. Therefore, the propylene oxide wastewater is diluted before treatment, and the wastewater can be treated after the concentration of chloride ions is reduced, so that a great deal of waste of dilution water is caused. Through screening salt-tolerant microorganisms in the natural environment, the effective degradation of the original COD of the epoxypropane saponification wastewater stock solution is realized by matching with the original activated sludge, the pressure of the downstream treatment process can be reduced, and the water treatment cost is further reduced.

The Klebsiella species belong to the genus Klebsiella of the family Enterobacteriaceae and are gram-negative bacteria. Under the optical microscope, the bacillus is short and thick, the size is about 0.5-0.8 multiplied by 1-2 μm, and the bacillus is singly arranged in a double or short chain shape. There are no spores, no flagella, thicker capsule, and most of them have pili. Several strains of the Klebsiella are important conditional pathogenic bacteria and iatrogenic infectious bacteria, wherein the Klebsiella pneumoniae (Klebsiella peneumoniae) has the strongest pathogenicity to people, but researches on agricultural microorganisms, industrial pollution treatment and the like show that the Klebsiella is not only a green bacterial fertilizer, but also a high-efficiency purifying agent. Klebsiella variicola (Klebsiella variicola) is an important member of the genus Klebsiella, and has been reported in documents and patents to have biological control and biosynthesis application values (for example: Zhao Mingfu et al, the dynamic colonization of Klebsiella variicola in dendrobe and the prevention effect test on dendrobe black spot, university of southwest forestry, 2015. Chinese patent document CN107384975A), but has not been reported in the treatment of high-salt wastewater, especially propylene oxide saponification wastewater.

Chinese patent document CN110106123A (application number: 201910466823.6) discloses a Klebsiella strain Z3 and application thereof in reduction of hexavalent chromium ions, the disclosed Klebsiella strain Z3 can tolerate 60g/L NaCl at most, while the Klebsiella variicola related to the invention can tolerate 90g/L NaCl, which is obviously superior to the salt tolerance of the Klebsiella strain Z3.

Propylene oxide is an important chemical raw material, is used for producing propylene glycol, polyether, surfactant and the like in a large amount, and is the third largest propylene derivative which is second only to polypropylene and acrylonitrile. Currently, 8% of the world's propylene consumption is used for propylene oxide production. The propylene oxide production line of domestic enterprises at present adopts a chlorohydrination method production process, wastewater produced by the process has the characteristics of high temperature, high pH value, high chlorine root content, high COD content and high suspended matter content, the inlet water temperature of the wastewater is generally 30-40 ℃, the pH value is 9-11, CaCl is added₂The concentration is about 3.5-4.0%, the COD is about 1000mg/L, and the chloride ion concentration is about 23000-25000 mg/L. The complexity of the components determines that the waste water is difficult to treat, about 50 tons of saponified waste water is generated every 1 ton of propylene oxide is produced, the waste water amount is very large, and therefore how to effectively treat COD in the waste water is the biggest difficulty for environmental protection treatment of companies and the problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides Klebsiella variicola and application thereof.

The Klebsiella variicola provided by the invention has the effect of efficiently removing COD in the epoxypropane saponified sewage, and the removal rate of the COD is 81.31%.

Aiming at the treatment of the epoxypropane saponification wastewater, the Klebsiella variicola is obtained by screening the activated sludge of the epoxypropane saponification wastewater, and the Klebsiella variicola is detected to have the characteristic of efficiently degrading the COD of the epoxypropane saponification wastewater, can be used as an additive of the normal epoxypropane saponification wastewater sludge, and improves the treatment effect.

The propylene oxide saponification wastewater refers to wastewater generated in the production of propylene oxide by a chlorohydrin method.

The technical scheme of the invention is as follows:

klebsiella variicola (Klebsiella variicola) KV-8, deposited at 7/1/2021 in the China center for type culture Collection at the following addresses: the preservation number of the eight-path Lojia mountain Wuhan university in Wuchang district, Wuhan city, Hubei province is CCTCC NO: m2021023.

Klebsiella variicola (Klebsiella variicola) KV-8 is hereinafter referred to as "Klebsiella variicola KV-8".

The shape characteristics of the Klebsiella variicola KV-8 are as follows: as shown in fig. 1, klebsiella belongs to the genus klebsiella of the family enterobacteriaceae, which is a gram-negative bacterium; short and thick bacilli under an optical microscope, the size of the bacilli is about 0.5-0.8 multiplied by 1-2 mu m, and the bacilli are singly arranged in a double or short chain shape; there are no spores, no flagella, thicker capsule, and most of them have pili.

The preparation method of the Klebsiella variicola KV-8 comprises the following steps:

inoculating Klebsiella variicola KV-8 on a solid culture medium, and performing inversion activation culture at 25-37 deg.C for 40-50h to obtain activated strain; inoculating the activated strain into a liquid culture medium, and culturing at 25-37 deg.C and 150-180rpm for 20-40h to obtain a seed solution; transferring the prepared seed liquid into a liquid culture medium B according to the volume percentage of 1-3%, and carrying out amplification culture at the temperature of 25-37 ℃ and the rotational speed of 180rpm for 20-40h to obtain the Klebsiella variicola KV-8 bacterial liquid.

According to a preferred embodiment of the present invention, in the above preparation method, the solid medium is an LB solid medium, and the liquid medium is an LB liquid medium.

The Klebsiella variicola KV-8 can be used for treating wastewater.

According to the invention, the Klebsiella variicola KV-8 is preferably used for treating high-chlorine-containing wastewater.

Further preferably, the Klebsiella variicola KV-8 is used for treating wastewater containing high chlorine, high salt and alkaline.

Further preferably, the Klebsiella variicola KV-8 is applied to the treatment of wastewater with salinity (W/V) of 9% or less.

Further preferably, the Klebsiella variicola KV-8 is used for treating the propylene oxide saponification wastewater.

Advantageous technical effects

The Klebsiella variicola KV-8 has the characteristics of salt resistance, alkali resistance, high chlorine resistance and the like, and the epoxypropane saponified sewage activated sludge added with the Klebsiella variicola KV-8 has obvious effect of improving COD removal, the removal rate is 81.31 percent, and the removal rate of 48 hours is 12.7 mg/(L.h).

Drawings

FIG. 1 is a gram-stained image of Klebsiella variicola KV-8;

FIG. 2 is a phylogenetic tree of Klebsiella variicola KV-8;

FIG. 3 is a graph showing the growth of Klebsiella variicola KV-8 under different salinity conditions;

FIG. 4 is a graph showing the growth of Klebsiella variicola KV-8 under different pH conditions;

FIG. 5 is a graph showing the effect of COD treatment on the propylene oxide saponified wastewater by the activated sludge of the propylene oxide saponified wastewater inoculated with Klebsiella variicola KV-8.

Detailed Description

The invention is further illustrated with reference to specific examples, without however restricting the scope of the invention thereto.

The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by manufacturers, and are all common commercial products.

Example 1

Screening Klebsiella variicola KV-8 comprises the following steps:

the strain sieve is selected from residual sludge of epoxy propane saponification wastewater, 1mL of residual sludge sample is taken and placed in a 150mL sterile triangular flask, 50mL of sterile physiological saline and 2g of sterile glass beads are added, the mixture is shaken for 15 minutes at 25 ℃ and 180rpm, the bacterial liquid is absorbed and is subjected to gradient dilution by sterile water until the bacterial liquid is respectively diluted to 10^-2、10^-3、10^-4、10^-5、10^-6And (2) respectively sucking 100 mu L of diluent, coating the diluent on a sterile LB solid medium plate containing 5%, 8% and 10% of sodium chloride by mass concentration, standing and culturing for 2d at 25 ℃, picking up a clone which grows fast and has large colonies to 50mL of LB liquid medium containing 10% of sodium chloride by mass concentration, culturing for 2d at 25 ℃ and 180rpm, sucking 100 mu L of bacterial liquid, coating the bacterial liquid on a sterile LB solid medium containing 10% of sodium chloride by mass concentration, and picking up a single clone.

The 16S rDNA sequence was obtained by PCR amplification using a monoclonal lawn as a template and 27F (AGA GTT TGA TCC TGG CTC AG SEQ ID NO.2) and 1492R (TAC GGC TAC CTT GTT ACG ACT T SEQ ID NO.3) as primers. The PCR reaction was as follows (50. mu.L), see Table 1:

TABLE 1

Colony PCR program parameters were set as follows:

the PCR product is recovered by an Omega gel recovery kit and then sent to Qingdao catalpi-xi biotechnology limited company for sequencing, the 16S rDNA sequence contains 1468bp, the nucleotide sequence is shown as SEQ ID NO.1, the sequence is compared with BLAS (website: http:// blast.ncbi.nlm.nih.gov/blast.cgi) in GenBank database, and a phylogenetic tree is constructed, as shown in figure 2, the result shows that the bacterium belongs to Klebsiella variicola and is named as Klebsiella variegata KV-8.

Klebsiella variicola (Klebsiella variicola) KV-8, deposited at 7 days 1 month in 2021 at the China center for type culture Collection with the following addresses: the preservation number of the eight-path Lojia mountain Wuhan university in Wuchang district, Wuhan city, Hubei province is CCTCC NO: m2021023.

Example 2

The fermentation culture method of Klebsiella variicola KV-8 comprises the following steps:

taking Klebsiella variicola KV-8, streaking on LB solid medium, inverting at 25 deg.C, activating and culturing for 2 days to obtain activated strain; inoculating the activated strain into LB liquid culture medium, and shake culturing at 25 deg.C and 180rpm for 1 day to obtain seed solution; transferring the prepared seed liquid into an LB liquid culture medium according to the volume percentage of 1 percent, and carrying out amplification culture at 25 ℃ and 180rpm for 1 day to obtain a bacterial liquid; the bacterial concentration of the bacterial liquid is 5 multiplied by 10 after detection⁹cfu/mL。

The liquid culture medium is LB liquid culture medium, and the components are as follows: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, and the volume is constant to 1L, and the pH is natural.

The solid culture medium is LB solid culture medium, and the components are as follows: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar powder, wherein the volume is constant to 1L, and the pH is natural.

Examples of the experiments

(1) The growth conditions of Klebsiella variicola KV-8 under different salinity conditions are as follows:

inoculating 0.1mL of Klebsiella variicola KV-8 into an LB culture medium, and culturing at 25 ℃ and 200rpm for 24h to obtain activated bacterial liquid; inoculating the prepared activated bacterial liquid into LB liquid culture medium with different salinity (w/v) of 1%, 3%, 5%, 7%, 9%, 11% and 15% according to the volume percentage of 1%, performing shake culture at 25 ℃ and 200rpm, sampling once at intervals of 6h, and determining the OD of the bacterial liquid₆₀₀The strain growth curve was plotted, and the results are shown in FIG. 3.

LB liquid culture media with different salinity comprise the following components: 10g of peptone, 5g of yeast powder and 5g of sodium chloride, wherein the amount of sodium chloride is calculated according to salinity (w/v) is 1%, 3%, 5%, 7%, 9%, 11% and 15%), the volume is adjusted to 1L, and the pH is natural.

(2) The growth conditions of Klebsiella variicola KV-8 under different pH conditions are as follows:

inoculating 0.1mL of Klebsiella variicola KV-8 into an LB culture medium, and culturing at 25 ℃ and 200rpm for 24h to obtain activated bacterial liquid; inoculating the activated bacteria liquid into LB liquid culture medium with different pH values according to volume percentage of 1%, shake culturing at 25 deg.C and 200rpm with pH values of 6, 7, 8, 9, 10 and 11, sampling once at interval of 6h, and measuring OD of bacteria liquid₆₀₀The strain growth curve was plotted, and the results are shown in FIG. 4.

LB liquid culture medium with different pH values comprises the following components: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, wherein the constant volume is 1L, and the pH values are respectively adjusted to 6, 7, 8, 9, 10 and 11.

According to the experimental results, the Klebsiella variicola KV-8 disclosed by the invention can tolerate 9% of salinity and can grow well when the salinity is lower than 9%. In addition, the bacterium can adapt to neutral and strong alkaline environment, and can grow well even when the pH is 9. The bacteria can grow well in a high-salt and strong-alkaline environment, and the application of the bacteria in the treatment of the propylene oxide saponification wastewater has great advantages.

Application example

Experiment for sewage treatment by using Klebsiella variicola KV-8 and experiment effect

Culturing Klebsiella variicola KV-8 in LB liquid culture medium for 24h, centrifuging the cultured bacteria liquid at 10000rpm for 10min, and pouring off the supernatant to obtain the thallus at the bottom of the centrifuge tube. And then reselecting the thallus by using 5mL of physiological saline, centrifuging for 10min at 10000rpm, and repeating twice to eliminate the influence of organic matters brought by the LB liquid culture medium in the bacterial liquid on the sewage COD. Finally, the obtained bacterial suspension is inoculated into a conical flask filled with 200mL of propylene oxide saponified sewage and propylene oxide saponified activated sludge (MLSS is about 30g/L) with the volume fraction of 10%, the mixture is cultured at 25 ℃ and 200rpm, samples are respectively taken for 0h, 12h, 24h, 36h and 48h after the inoculation of Klebsiella variicola KV-8 to determine the removal effect of COD by the propylene oxide saponified sewage activated sludge, and the results are shown in figure 5 compared with the treatment effect of the propylene oxide saponified sewage activated sludge without the inoculation of Klebsiella variicola KV-8. As can be seen from the figure, the removal rate of COD by the activated sludge of the saponified sewage of propylene oxide which is not inoculated with Klebsiella variicola KV-8 within 48h is only 69.76%, while the removal rate of COD by the activated sludge of the saponified sewage of propylene oxide which is inoculated with the Klebsiella variicola KV-8 within 48h is 12.7 mg/(L.h), the removal rate of COD is 81.31%, the removal rate of COD in the sewage is increased by 11.55%, and the removal effect is obviously improved. This shows that Klebsiella variicola KV-8 inoculated to the sludge can adapt to the environments of high salt, high chlorine, high alkali and high COD of the propylene oxide saponified sewage, and can grow rapidly to degrade organic matters in the propylene oxide saponified sewage, thereby effectively reducing the COD of the propylene oxide saponified sewage.

The waste water produced by producing the propylene oxide by the chlorohydrination method contains organic chloride with certain concentration besides the characteristics of high salt, high chlorine and high pH. Organic chlorides have been used as highly effective insecticides, such as heptachlorobicycloheptadiene, hexachloro cyclohexane, dichlorodiphenyl cyclohexane, hexachloro benzene, etc., because of their high stability and high efficiency in removing crop pests. These organic chlorides have a lethal effect not only on insects, but also on the growth and normal metabolism of microorganisms. The Klebsiella variicola KV-8 overcomes the factors unfavorable for microbial metabolism, and can effectively reduce COD of the epoxypropane saponified sewage.

SEQUENCE LISTING

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Claims

1. Klebsiella variicola (Klebsiella variicola) KV-8, deposited at 7/1/2021 in the China center for type culture Collection at the following addresses: the preservation number of the eight-path Lojia mountain Wuhan university in Wuchang district, Wuhan city, Hubei province is CCTCC NO: m2021023.

2. A process for producing Klebsiella variegata (Klebsiella variicola) KV-8 according to claim 1, characterized by comprising the steps of:

inoculating Klebsiella variicola KV-8 on a solid culture medium, and performing inversion activation culture at 25-37 deg.C for 40-50h to obtain activated strain; inoculating the activated strain into a liquid culture medium, and culturing at 25-37 deg.C and 150-180rpm for 20-40h to obtain a seed solution; transferring the prepared seed liquid into a liquid culture medium according to the volume percentage of 1-3%, and carrying out amplification culture at the temperature of 25-37 ℃ and the rotational speed of 180rpm for 20-40h to obtain the Klebsiella variicola KV-8 bacterial liquid.

3. The method for producing Klebsiella variicola (Klebsiella variicola) KV-8 as claimed in claim 2, wherein the solid medium is LB solid medium and the liquid medium is LB liquid medium.

4. Use of Klebsiella variicola (Klebsiella variicola) KV-8 as claimed in claim 1 in the treatment of chlorine-containing waste water.

5. Use according to claim 4, wherein said Klebsiella variicola (Klebsiella variicola) KV-8 is used in the treatment of chlorine-, salt-, and alkaline waste water.

6. Use according to claim 5, wherein the Klebsiella variicola (Klebsiella variicola) KV-8 is used in the treatment of waste water having a salinity (W/V) of less than 9%.

7. Use according to claim 5, wherein said Klebsiella variicola (Klebsiella variicola) KV-8 is used in the treatment of saponified wastewater with propylene oxide.