Method for preparing kaolin immobilized GY2B bacteria and application thereof

Patent metadata

Field	Value
Publication number	US-10174306-B2
Application number	US-201415507599-A
Country	US
Kind code	B2
Filing date	Dec 3, 2014
Priority date	Aug 29, 2014
Publication date	Jan 8, 2019
Grant date	Jan 8, 2019

Abstract

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

First claim

The invention claimed is:

1. A method for preparing a composition comprising kaolin-immobilized Sphingomonas GY2B (GY2B) bacteria, comprising the steps of:
(a) activating and growing a GY2B bacterial culture by culturing the GY2B bacteria in a phenol-containing enrichment medium to obtain a concentrated bacterial suspension;
(b) centrifuging the concentrated bacterial culture;
(c) washing the concentrated bacterial culture with a sterile physiological saline solution;
(d) suspending the washed concentrated bacterial culture in a sterile physiological saline solution to obtain a bacterial suspension;
(e) adding to the bacterial suspension a sterilized kaolin suspension comprising kaolin dissolved in a sterile MSM culture solution in an inoculation amount of 2-8%; and
(f) incubating the preparation made in step (e) for 1-4 hours to adsorb and immobilize the GY2B bacteria on the kaolin, thereby preparing kaolin-immobilized GY2B bacteria.

2. The method according to claim 1 , wherein the activation and growth of the GY2B bacteria in claim 1 , step (a), is carried out as follows:
(a) picking 1-2 GY2B colonies stored on a plate and inoculating it/them into a sterile MSM culture solution containing 50-200 mg/L phenol,
(b) shaking the culture medium at 25-35° C. 100-200 rpm, in the dark, for 8-16 hours,
(c) then removing 1-2 mL of the bacterial culture and subculturing in a fresh sterile MSM culture solution containing 50-200 mg/L phenol, for 8-16 hours, and
(d) repeating the subculturing step of step (c) up to six times, to obtain GY2B bacteria having a stable ability to degrade phenol, and
(e) plating and storing the GY2B bacteria made in step (d).

3. The method according to claim 1 , wherein, in step (a), the phenol concentration in the medium is 50-200 mg/L, and wherein the GY2B bacteria are cultured for 8-16 hours.

4. The method according to claim 1 , wherein the bacterial suspension obtained in step (a) is adjusted to have an absorbance at 600 nm (OD 600 ) of 0.8-1.5.

5. The method according to claim 1 , wherein the centrifugation and washing steps are carried out at a temperature of 2-10° C., the centrifugation speed ranges from 6000-12,000 rpm, the centrifugation time ranges from 10-20 min. and the number of times for the centrifugation and washing steps ranges from 2-5.

6. A method according to claim 1 , wherein the sterilization of the kaolin comprises:
(a) autoclaving the kaolin at a temperature ranging from 100-150° C., for a time ranging from 15-60 min.,
(b) drying the kaolin at a temperature of 80-150° C. for 10-30 min., and
(c) dispersing the kaolin.

7. The method according to claim 1 , wherein the kaolin has a mesh size of 100-300, and wherein step (f) is carried out in a constant-temperature shaking incubator at a speed of 100-200 rpm, at a temperature of 25-35° C., in the dark.

Assignees

• Univ South China Tech

Inventors

• Wu Pingxiao
• Li Yuewu
• Dang Zhi
• Zhu Nengwu

Classifications

• C12N5/00

  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) · CPC title

• C12N11/02

  Enzymes or microbial cells immobilised on or in an organic carrier · CPC title

• C12N11/14 Primary

  Enzymes or microbial cells immobilised on or in an inorganic carrier · CPC title

• C12R1/00

  Chemistry & Metallurgy · mapped topic

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title