A single-cell oil-producing cryptococcus and a method for producing oil by culturing crude glycerol

A single-cell oil and cryptococcus technology, applied in the field of microorganisms, can solve the problems of complex composition, staying in the laboratory research stage, and unstable components.

Active Publication Date: 2021-05-18
WENZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There are many reports at home and abroad on the research on the production of single-cell oil by culturing Cryptococcus, especially in order to reduce costs, a large number of studies have been carried out on suitable raw materials for the production of single-cell oil by culture of Cryptococcus, including the metabolite acetic acid in the anaerobic fermentation process (G. Christophe, et al. Biochem Biotechnol, 2012, 167: 1270-1279), cheese whey, a by-product of the cheese manufacturing process (Yeong Hwan Seo, et al. Biochemical Engineering Journal, 2014, 90: 149-153), Corn cob hydrolyzate (Yi-Huang Chang, et al. Fuel, 2013, 105:711-717.), cellulose hydrolyzate (Zhang Jie, et al. Modern Chemicals, 2008, 28(2): 133-135), hydrolysis Food waste and municipal sewage (ZhanyouChi et al.Biochem Biotechnol,2011,165:442-453.), organic wastewater from brewing industry (Byung-GonRyu.Bioresource Technology,2013,135:357-367), etc., of course, Due to the complex composition and unstable composition of these industrial and agricultural wastes, the cultivation of cryptococcus to produce single-cell oil only stays in the laboratory research stage, and it is difficult to realize large-scale production for a while.

Method used

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  • A single-cell oil-producing cryptococcus and a method for producing oil by culturing crude glycerol
  • A single-cell oil-producing cryptococcus and a method for producing oil by culturing crude glycerol
  • A single-cell oil-producing cryptococcus and a method for producing oil by culturing crude glycerol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Isolation and identification of Cryptococcus curvatus WZUC01

[0041] (1) Primary screening

[0042] Samples were collected from the Ou Tangerine Garden in Chashan Town, Wenzhou City, Zhejiang Province. The samples included soil, dried cow dung, and slightly rotten Ou Tangerine. The soil samples were collected at a depth of 0-10 cm, and the sampling volume at each point was greater than 100 g. Weigh 10g of each sample (slightly rotten mandarin orange peel and weigh it and crush it) and place it in a Erlenmeyer flask filled with 90ml of sterile water, shake it up and dilute to an appropriate multiple, take a drop and spread it on a YPD plate (glucose 20g / L, yeast powder 10g / L, peptone 20g / L, agar 18g / L, pH natural), culture at 30°C until a single colony grows; pick a single colony that is not mold and inoculate it on a new YPD plate. Cultivate at the same temperature until it is a pure culture by microscopic examination. A total of 105 pure cultures were ob...

Embodiment 2

[0055] Embodiment two: the pretreatment of biodiesel by-product crude glycerin

[0056]Crude glycerin, a by-product of biodiesel, is obtained from biodiesel production enterprises, and is used to produce biodiesel from kitchen waste oil.

[0057] After mixing crude glycerol, a by-product of biodiesel, with water at a ratio of 1:4 (V:V), adjust the pH to 6.5, fatty acids form fatty acid salt (soap) precipitation, centrifuge at 4000 rpm for 10 minutes to remove soap to obtain crude glycerin, and then Sterilize with high pressure steam for 10 minutes (to be sterilized after the medium is prepared when it is used to prepare the medium in the future) to remove methanol.

[0058] The pretreated crude glycerin was analyzed for relevant components, and the results are shown in Table 2. Minerals were measured by atomic absorption spectrophotometry, glycerol content was measured by potassium periodate method (Li Sumin, Chinese Journal of Blood Transfusion, 1999, 12(3):179~179), fatty a...

Embodiment 3

[0061] Embodiment three: the determination of Cryptococcus curvatus WZUC01 seed growth curve

[0062] Preserved strain WZUC01 (1.5ml frozen tube thawed bacteria solution) was inoculated in 50ml seed medium (20g / L crude glycerol, 10g / L yeast powder, 20g / L peptone, natural pH, crude glycerol refers to pretreated crude The amount of glycerol contained in glycerin (hereinafter the same)) was placed in a 250ml Erlenmeyer flask, and activated at 30°C and 150r / min for 48-72h. The activated bacteria were inoculated in a 500ml Erlenmeyer flask containing 100ml seed culture medium (crude glycerol 20g / L, yeast powder 10g / L, peptone 20g / L, natural pH) with a volume ratio of 5%. Cultivate at 150r / min, take samples regularly to measure the biomass (OD 600 ), with time versus lnOD 600 The growth curve of Cryptococcus curvatus WZUC01 is drawn as follows image 3 . Depend on image 3 It can be seen that Cryptococcus curvatus WZUC01 enters the stationary phase after 31.5 hours of culture, ...

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Abstract

The invention discloses a single-cell oil-producing cryptococcus and a method for cultivating the single-cell oil-producing cryptococcus with crude glycerol, a by-product of biodiesel. The bacterial strain is Cryptococcus curvatus (Cryptococcus curvatus) WZUC01, which is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the registration number of the preservation center is CGMCC NO.8096. The strain has high oil content and the main fatty acids are palmitic acid, stearic acid and oleic acid, and its fatty acid composition is suitable for biodiesel production. The fed-batch culture method was used to culture Cryptococcus curvatus WZUC01 with the pretreated biodiesel by-product crude glycerol. After 160 hours of culture, the biomass and oil production reached 81.37±2.01g / L and 66.89±3.15g / L, respectively, which has a great effect. potential for practical application.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a cryptococcus strain producing single-cell oil and a method for cultivating the cryptococcus to produce single-cell oil with crude glycerol, a by-product of biodiesel. Background technique [0002] In recent years, with the rapid development of economy and science and technology, people's demand for coal, oil and other fuels has been increasing. However, the excessive consumption of these fuels has caused global problems such as environmental degradation and tightening of energy resources, which has prompted people to start Looking for green and renewable alternative fuels, biodiesel began to enter the field of people's life and developed rapidly. Although my country's biodiesel production started relatively late, it will reach 2.27 billion liters by 2020 in accordance with the Medium and Long-Term Renewable Energy Development Plan of Sinopec, PetroChina and CNOOC. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/64C12P7/20C12R1/01
CPCC12P7/20C12P7/6463C12P7/649C12N1/205C12R2001/01Y02E50/10
Inventor 周茂洪赵肖为
Owner WENZHOU UNIV
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