# US 2017/0283786 A1 · Method for preparing kaolin immobilized gy2b bacteria and application thereof

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- Number: US 2017/0283786 A1
- Document ID: US20170283786A1
- Status: Published application
- Kind: A1
- Application number: 15/507,599
- Filed: 2014-12-03
- Published: 2017-10-05
- Earliest claimed: 2014-08-29
- Assignees: South Cina University of Technology
- Applicants: South China University of Technology
- Inventors: Pingxiao WU; YUEWU LI; ZHI DANG; NENGWU ZHU
- Web page: https://patentgrep.com/patent/US20170283786A1

## CPC

- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C02F 3/34: characterised by the microorganisms used
- C12N 1/20: Bacteria; Culture media therefor
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 11/02: Enzymes or microbial cells immobilised on or in an organic carrier
- C12N 11/12: Cellulose or derivatives thereof
- C02F 2101/345: {Phenols}
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12R 2001/00: Microorganisms {; Processes using microorganisms}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

Provided are a method for preparing kaolin immobilized GY2B bacteria and use thereof.

## Claims

### Claim 1

A method for preparing immobilized GY2B bacteria, comprising the steps of:
activating a GY2B bacteria culture: growing the activated GY2B bacterial culture in a phenol-containing enrichment culture solution, so as to obtain a concentrated bacterial solution centrifuging the concentrated bacterial culture; washing the concentrated bacterial culture with a sterile physiological saline; suspend the washed concentrated bacterial culture in a sterile physiological saline so as to obtain a bacterial suspension;
adding to the bacterial suspension a sterilized kaolin suspension comprising kaolin dissolved in a sterile MSM culture solution. in a inoculation amount of 2˜8%, adsorbed and immobilized for 1˜4 hr, so as to prepare the kaolin immobilized GY2B bacteria.

### Claim 2 (depends on 1)

A method according to claim 1, wherein the activation and cultivation of the GY2B bacteria in claim 1 is carried out as follows: 1˜2 GY2B colonies stored in a plate are picked into a sterile MSM culture solution containing 50˜200 mg/L phenol, shaken and cultured at 25˜35° C., 100˜200 r/min, in dark, for 8˜16 hr, then 1˜2 mL of the bacterial solution is taken, sub-cultured in a fresh sterile MSM culture solution containing 50˜200mg/L phenol, for 8˜16 hr, then 1˜2 ml of the well grown bacterial solution is taken, and sub-cultured in a fresh sterile MSM culture solution containing 50˜200mg/L phenol, and repeated the sub-culturing as such 2-5 times, so as to obtain a GY2B bacteria having an ability to stably degrade phenol, which is plated and stored.

### Claim 3 (depends on 1)

A method according to claim 1, wherein the bacteria are grown in an enrichment culture solution containing 50˜200 mg/L phenol for 8˜16 h so as to obtain the concentrated bacterial solution.

### Claim 4 (depends on 1)

A method according to claim 1, wherein the concentration of the bacterial suspension obtained is adjusted in an ultraviolet spectrophotometer at 600 nm to make the absorbance of A=0.8˜1.5.

### Claim 5 (depends on 1)

A method according to claim 1, wherein the centrifugation and washing further comprises a centrifugation temperature ranging from is 2-10° C., the centrifugation speed is ranging from 6000˜12000 r/min, the centrifugation time ranging from 10-20 min, and the number of centrifugation and washing is ranging from 2-5 times.

### Claim 6 (depends on 1)

A method according to claim 1, wherein the sterilization of kaolin comprises: the kaolin is autoclaved at a temperature ranging from 100˜150° C., for a time ranging from 15˜60 min, then dried and dispersed at a temperature ranging from 80˜150° C. for a time ranging from 10˜30 min.

### Claim 7 (depends on 1)

A method according to claim 1, wherein the kaolin is of 100-300 meshes, the adsorption and immobilization in step (2) are carried out in a constant-temperature shaking incubator at a rate ranging from 100˜200 r/min, 25˜35° C., in dark.

### Claim 8 (depends on 1)

An immobilized bacteria, comprising a kaolin immobilized GY2B bacteria obtained by the method according to claim 1.

### Claim 9 (depends on 1)

A method of treating phenol-containing waste water, comprising treating the phenol-containing waste water with an amount of a kaolin immobilized GY2B bacteria prepared according to claim 1.

### Claim 10 (depends on 9)

The method of claim 9, wherein the kaolin immobilized GY2B bacteria comprise a kaolin concentration in a range of 15˜30 g/L, a pH ranging from 7˜12, and a phenol concentration ranging from 0˜300 mg/L.