CN102250974A - Preparation method of microbial oil - Google Patents

Abstract

The invention discloses a method for preparing microbial oil. The method comprises the following steps of: performing aerated culturing on a carbon source which contains glucose and xylose and serves as a raw material to obtain a leavening; and extracting the microbial oil from the leavening. According to the method, glucose and xylose in a culture medium can be transformed synchronously, so that the inhibition function of the glucose on the utilization of other substrates during the utilization of mixed sugar is relieved, the phenomenon of secondary growth in the process is avoided, the substrate transformation rate is high, and the production cost can be lowered remarkably. The method has a great application prospect in the aspect of the production of microbial oil with a lignocellulose material.

Description

A kind of preparation method of microbial grease

technical field

The invention belongs to the production of microbial oil, in particular to a method for preparing microbial oil by utilizing oil-producing microorganisms to convert carbon sources containing glucose and xylose.

Background technique

Microbial oils, also known as single-cell oils, are microorganisms such as yeast, mold, bacteria, and algae that convert carbohydrates into oils and accumulate in cells under certain conditions. Any microorganism that can accumulate oil in cells exceeding 20% by weight of the dry weight of the cell is called an oleaginous microorganism. According to reports, some microorganisms accumulate oil to reach more than 70% of the dry weight of the cell (Li Yonghong, Liu Bo, Zhao Zongbao, Bai Fengwu. Research on the optimization of the fermentation medium and fermentation conditions of Rhodosporidium toruloides for oil production. Chinese Journal of Bioengineering, 2006, 22 (4): 650-656). Compared with the production of oil plants, microbial conversion of carbohydrates to produce oil is not affected by site, season and climate changes, and large-scale continuous production of oil can be realized. The composition of biodiesel produced by transesterification of microbial oil is similar to that of animal and vegetable oil. Therefore, microbial oil technology can provide new raw materials for the development of bioenergy industry and has important application prospects.

Lignocellulosic materials are low-cost, widely sourced, and abundant in total resources, and their chemical components are mainly cellulose, hemicellulose, and lignin. The main product of cellulose hydrolysis is glucose; the hydrolysis of hemicellulose produces a mixture of glucose, xylose, galactose, arabinose, and mannose. If oleaginous microorganisms can be cultivated using lignocellulose hydrolyzate as a carbon source, the cost of microbial oil production will be significantly reduced.

When microorganisms use the mixture of hexose and pentose sugar, most microorganisms use glucose first, and start to use other carbon sources when glucose is exhausted or at a very low concentration. This phenomenon is called carbon source metabolic repression, or the glucose effect (Goerke B, Stulke J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol, 2008, 6(8): 613-624; Nichols N N, Dien B S, Bothast R J.Use of cataboliterepression mutants for fermentation of sugar mixtures to ethanol. Appl Microbiol Biotechnol, 2001, 56(1-2): 120-125). According to literature reports, the oil-producing microorganism Lipomyces starkeyi (Kong Xiangli, Liu Bo, Zhao Zongbao, Feng Bin. Starkeyi uses mixed sugar fermentation to produce oil. Bioprocessing, 2007, 5 (2): 36- 41), Trichosporon fermentans (Huang C, Zong M, Wu H, Liu Q. Microbialoil production from rice straw hydrolysate by Trichosporon fermentans. Bioresour Technol, 2009, 100(19): 4535-4538) etc. are in use When a mixture of glucose and xylose is used, glucose is first utilized, and then xylose begins to be utilized. This leads to a lag period in the growth of microorganisms and production of products, ie secondary growth, which results in prolonged fermentation cycles and reduced efficiency. Therefore, finding microorganisms that can simultaneously and efficiently utilize hexoses and pentoses is of great significance for improving fermentation efficiency.

Relevant studies on releasing carbon source metabolic repression have been reported in the literature. In the continuous fermentation process, the glucose concentration was maintained at a low level, and the simultaneous utilization of glucose and xylose was realized at a more suitable dilution rate (Kastner J, Jones W, Roberts R.Simultaneous utilization of glucose and D -xylose by Candida shehatae in a chemostat. J Ind Microbiol Biot, 1998, 20(6): 339-343). Secondly, by inducing the enzymes required for xylose metabolism in the early stage, the simultaneous utilization of mixed sugars can also be tested (Kastner J, Roberts R.Simultaneousfermentation of D-xylose and glucose by Candida shehatae.Biotechnol Lett, 1990, 12(1): 57-60). In addition, by screening glucose phosphotransferase mutants, glucose inhibition can also be released, glucose and xylose can be utilized simultaneously, and secondary growth can be eliminated (Dien B, Nichols N, Bothast R. Fermentation of sugar mixtures using Escherichia colicatabolite repression mutants engineered for production of L-lactic acid. J Ind Microbiol Biot, 2002, 29(5): 221-227).

Contents of the invention

When the carbon source contains glucose and xylose, most oleaginous microorganisms use glucose preferentially, and only start to use xylose when glucose is completely consumed or the concentration drops to a very low level, which is called glucose inhibition, and the direct result is microbial growth and product A secondary growth phenomenon occurs during the accumulation process. The secondary growth phenomenon in the oil fermentation process leads to prolonged fermentation period, reduced efficiency and increased production cost. The inventors believe that eliminating the secondary growth phenomenon of oil production can significantly promote the effective utilization of cheap carbon sources and improve the technical economy of microbial oils. Therefore, the inventors conducted scientific experiments using a large number of oil-producing microorganisms, and found that some strains of oil-producing microorganisms have the physiological characteristics of synchronously consuming glucose and xylose.

The object of the present invention is to provide a method for synchronously utilizing carbon sources containing glucose and xylose to cultivate oil-producing microorganisms to produce microbial oils containing one or more fatty acids and derivatives thereof. The invention has simple process and easy method, can more efficiently utilize and transform carbon sources with complex components, such as hydrolyzate of lignocellulosic raw materials, and improves the technical economy of microbial oil production.

The present invention is achieved through the following technical solutions:

1. Preparation of oleaginous medium. Through one of the following methods or a necessary combination of them:

A. Mix glucose and xylose to make an aqueous solution with a total sugar concentration of 2% to 10%, add other necessary nutrients, pH 4.0 to 8.0, and sterilize for later use;

B. Hydrolyze the lignocellulosic raw material with reference to the literature method, the product is made into an aqueous solution containing glucose and xylose, and other necessary nutrients are added, the pH is 4.0-8.0, and it is sterilized for later use; or,

C. Refer to the literature method to hydrolyze the bio-based raw materials containing carbohydrates, add glucose or xylose and other necessary nutrients to the product, pH 4.0-8.0, and sterilize for later use.

Other necessary nutrients are: conventional substances used for the cultivation of oil-producing microorganisms, such as yeast powder and the like.

2) Cultivation of oil-producing microorganisms. Inoculate the oleaginous microorganism seed liquid into the medium described in step (1), the inoculum amount is 2% to 20% (v/v), and aerated culture at 25°C to 37°C until the residual sugar concentration in the mash is lower than 1 %.

3) Cell collection and microbial oil extraction. After the cultivation, the oleaginous microbial cells are concentrated and collected by centrifugation, filtration or other necessary methods. With reference to the literature (Li Zhifeng, Zhang Ling, Shen Xiaojing, etc. Comparative study of four kinds of fungal oil extraction methods. Microbiology Bulletin, 2001, 28 (6), 72-75) use acid heat-organic solvent method to extract oil, calculate Bacterial fat content.

The microbial oil can be further processed to make diesel fuel or a mixture used with diesel fuel.

The oleaginous microorganism used in the present invention is a fungus, bacterium or microalgae whose fat content in the thallus can exceed 20% (w/w) of the dry weight of the cell after fermentation and culture, and which has the physiological characteristics of synchronously consuming glucose and xylose. They include Trichosporon cutaneum, Cryptococcus curvatus, and Rhodosporidium toruloides. These strains can be purchased directly from strain collection institutions including China General Microorganism Culture Collection (CGMCC) and American Type Culture Collection (ATCC) or isolated from nature, or artificial or natural mutant strains.

The present invention adopts a method of mixing glucose and xylose, a method of hydrolyzing bio-based raw materials containing glucose units or xylose units, or a necessary combination method to prepare a carbon source containing glucose and xylose. Hydrolysis of bio-based raw materials refers to literature methods (Cara C, Ruiz E, Oliva J M, Saez F, Castro E. Conversion of olive tree biomass into fermentable sugars by diluteacid pretreatment and enzymatic saccharification. Bioresour Technol, 2008, 99(6): 1869 -1876) carried out. Lignocellulosic raw materials are materials containing cellulose, hemicellulose and lignin, including plants, crop straw and forestry processing waste, such as corn straw, wheat straw, straw, pine wood, spruce, etc.

The beneficial effects of the present invention are: 1) Compared with the traditional method for preparing biodiesel from animal and vegetable oils, the technology of the present invention is simple and effective, the cycle is short, and the cultivated land is saved; The inhibition of the utilization of other monosaccharides avoids the secondary growth phenomenon in the process and improves the efficiency of the fermentation process; 3) The microorganisms use glucose and xylose to accumulate microbial oils, which provides convenience for the efficient conversion of lignocellulosic raw materials to prepare microbial oils.

Description of drawings

The curves of substrate consumption and biomass production in Fig. 1 Example 1, wherein, ■glucose; ◆xylose; ▲biomass.

Detailed ways

Below is the specific embodiment that is prepared microbial grease by mixed sugar, can understand the influence that different processing conditions produce on microbial grease by embodiment.

Example 1

1) Mix glucose and xylose into a mixed sugar solution, glucose 47g/L, xylose 23g/L, add 1.0g/L yeast powder, 0.1g/L ammonium chloride, 1.0g/L magnesium chloride, 0.1g/L Sodium sulfate, 11.8g/L potassium dihydrogen phosphate, 3.7g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), the balance is water, pH 6.0, the obtained culture medium is sterilized at 121°C for 15 minutes for later use ;

2) Oleaginous yeast Trichosporon cutaneum AS 2.571 (purchased from China General Microorganism Culture Collection and Management Center) was cultured in a liquid seed medium at 30°C and 200 rpm for 20 hours with shaking;

3) Introduce the oleaginous microorganism seed solution prepared in step (2) into the medium described in step (1), inoculum size 10% (v/v), and aerobically culture at 30° C. for 120 h;

4) Terminate the fermentation. At this time, the residual glucose and xylose concentrations in the fermentation broth are respectively 0.1g/L and 1.9g/L; the solid-liquid separation collects the thalline and washes it twice with normal saline, and finally the dry thallus is 23.8g/L , the fat content is 49.7%.

Example 2

1) Mix glucose and xylose into a mixed sugar solution, glucose 10g/L, xylose 10g/L, add 2g/L yeast powder, 0.5g/L ammonium chloride, 0.2g/L magnesium chloride, 0.1g/L sulfuric acid Sodium, 0.4g/L potassium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), pH 5.0, and the obtained culture medium was sterilized at 121°C for 15 minutes for later use;

2) The oleaginous yeast Rhodosporidium toruloides AS 2.1389 (purchased from China Common Microorganism Culture Collection Management Center) was cultured in a liquid seed medium at 30°C and 200 rpm for 18 hours with shaking;

3) Introducing the oleaginous microorganism seed solution prepared in step (2) into the culture medium described in step (1), with an inoculum size of 2% (v/v), and culturing with aeration at 25°C for 60 hours;

4) Terminate the fermentation. At this time, the residual glucose and xylose concentrations in the fermentation broth were 0.1g/L and 1.2g/L respectively; solid-liquid separation collected the thalline and washed twice with normal saline, and dried to obtain 5.1g/L dry thallus. L; the oil content obtained by organic solvent extraction is 18%.

Example 3

1) Mix glucose and xylose into a mixed sugar solution, glucose 10g/L, xylose 50g/L, add 0.7g/L yeast powder, 0.2g/L ammonium chloride, 1.1g/L magnesium chloride, 0.5g/L Sodium sulfate, 8g/L potassium dihydrogen phosphate, 2g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), pH 6.5, the obtained culture medium was sterilized at 121°C for 15 minutes for later use;

2) Oleaginous yeast Cryptococcus curvatus ATCC 20509 (purchased from the American Type Culture Collection) was cultured in a liquid seed medium at 30°C and 200 rpm for 24 hours with shaking;

3) Introducing the oleaginous microorganism seed solution prepared in step (2) into the medium described in step (1), with an inoculum size of 5% (v/v), and aerated culture at 28°C for 144 hours;

4) termination of fermentation, now residual glucose and xylose concentrations in the fermented liquid were 0g/L, 6.6g/L; carry out post-treatment according to the method of Example 1, finally get dry thalline 15.7g/L, oil content 43.2 %.

Example 4

1) Mix glucose and xylose into a mixed sugar solution, glucose 30g/L, xylose 60g/L, add 0.3g/L yeast powder, 0.5g/L ammonium chloride, 1.2g/L magnesium chloride, 0.3g/L Sodium sulfate, 12g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), pH 7.0, the resulting culture medium was sterilized at 121°C for 15 minutes for later use;

2) Oleaginous yeast Cryptococcus curvatus ATCC 20509 (purchased from the American Type Culture Collection) was cultured in a liquid seed medium at 30°C and 200 rpm for 22 hours with shaking;

3) Add the oleaginous microorganism seed solution prepared in step (2) to the culture medium described in step (1), with an inoculum size of 15% (v/v), and culture in aeration at 32°C for 144 hours;

4) Terminate the fermentation. At this time, the residual glucose and xylose concentrations in the fermented liquid were 0.2g/L and 2.5g/L respectively; the post-treatment was carried out according to the method in Example 1, and finally the dried thallus was 26.9g/L, and the oil content was 26.9g/L. 48.6%.

Example 5

1) Mix glucose and xylose into a mixed sugar solution, glucose 95g/L, xylose 5g/L, add 1.2g/L yeast powder, 1.0g/L ammonium chloride, 0.6g/L magnesium chloride, 0.4g/L Sodium sulfate, 10g/L potassium dihydrogen phosphate, 6g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), pH 5.5, the resulting culture medium was sterilized at 121°C for 15 minutes for later use;

2) Oleaginous yeast Cryptococcus curvatus ATCC 20509 (purchased from the American Type Culture Collection) was cultured in a liquid seed medium at 30°C and 200 rpm for 28 hours with shaking;

3) Add the oleaginous microorganism seed liquid prepared in step (2) to the medium described in step (1), the inoculum size is 20% (v/v), and culture in aeration at 34°C for 168 hours;

4) Terminate the fermentation. At this moment, the residual glucose and xylose concentrations in the fermented liquid were 7.2g/L and 0g/L respectively; the post-treatment was carried out according to the method in Example 1, and finally the dried thallus was 24.3g/L, and the oil content was 40.1g/L. %.

Example 6

1) Mix glucose and xylose into a mixed sugar solution, glucose 20g/L, xylose 10g/L, add 0.2g/L yeast powder, 0.3g/L ammonium chloride, 0.8g/L magnesium chloride, 0.1g/L Sodium sulfate, 6g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, trace element solution 1% (v/v), pH 6.2, the obtained culture medium was sterilized at 121°C for 15 minutes for later use;

2) The oleaginous yeast Trichosporon cutaneum AS 2.571 (purchased from China Common Microorganism Culture Collection Management Center) was cultured in a liquid seed medium at 30°C and 200 rpm for 26 hours with shaking;

3) Add the oleaginous microorganism seed solution prepared in step (2) to the culture medium described in step (1), with an inoculum size of 12% (v/v), and culture in aeration at 37°C for 72 hours;

4) Terminate the fermentation. At this moment, the residual glucose and xylose concentrations in the fermented liquid were 0.5g/L and 2.1g/L respectively; the post-treatment was carried out according to the method in Example 1, and finally the dried thalline was 6.6g/L, and the oil content was 0.5g/L. 23.5%.

Example 7

Referring to literature method (Kootstra A, Beeffink H, Scott E, Sanders J.Comparison of dilute mineral and organic acid prevention for enzymatic hydrolysis of wheatstraw. Biochem Eng J, 2009, 46(2): 126-131), corn stalks were used as raw materials , dried, crushed through a 60-mesh sieve, mixed with 50mM maleic acid solution at a solid-liquid ratio of 10% (w/w), soaked for 24h, digested at 170°C for 30min, and diluted to a solid-liquid ratio of 5% (w/w) , adjust the pH to 4.8, the amount of cellulase added is 50FPU/g solid, react at 60°C and 150rpm for 72h, filter, adjust the total reducing sugar concentration to 25g/L, of which glucose is 17g/L and xylose is 8g/L. Add glucose 50g/L, 1.0g/L yeast powder, 0.6g/L ammonium chloride, 0.8g/L magnesium chloride, 0.1g/L sodium sulfate, 5g/L potassium dihydrogen phosphate, 2g/L Dipotassium hydrogen phosphate, 1% (v/v) trace element solution to adjust the pH to 5.0. After sterilizing at 121°C for 15 minutes, inoculate Trichosporon cutaneum AS 2.571 (purchased from China’s common microbial strains) with 10% (v/v) inoculum Preservation Management Center) seed liquid, and cultivated in aeration at 35°C for 144 hours. Fermentation was terminated. At this time, the residual glucose and xylose concentrations in the fermentation broth were 2.1 g/L and 0.2 g/L respectively. Post-treatment was carried out according to the method of Example 1, and finally 22.4 g/L of dry thalline was obtained, and the oil content was 38.5%.

Example 8

Referring to the literature (Sassner P, Martensson C G, Galbe M, Zacchi G.Steampretreatment of H2SO4-impregnated Salix for the production of bioethanol.Bioresour Technol, 2008, 99 (1): 137-145) method, with willow as raw material, dry , crushed through a 60-mesh sieve, soaked in excess 0.5% sulfuric acid for 90 minutes; filtered to obtain a solid content of 40%, stayed at 200°C for 8 minutes, and then carried out steam explosion; filtered, diluted with water to a solid content of 2 %, add cellulase 65FPU/g and glucosidase 393IU/g, react at 40°C for 96h; filter, adjust the total reducing sugar concentration to 60g/L, wherein glucose 45g/L, xylose 15g/L. Add 1.5g/L yeast powder, 1.0g/L ammonium chloride, 0.5g/L magnesium chloride, 0.1g/L sodium sulfate, 7.5g/L potassium dihydrogen phosphate, 2.5g/L dihydrogen phosphate to this sugar solution Potassium, trace element solution 1% (v/v) to adjust the pH to 6.0. After sterilizing at 121°C for 15 minutes, inoculate Trichosporoncutaneum AS 2.571 seed solution with 15% (v/v) inoculum, and incubate at 32°C for 100 hours. Fermentation was terminated, and the residual glucose and xylose concentrations in the fermentation broth were 0.8 g/L and 0.1 g/L respectively. Carry out aftertreatment according to the method of embodiment 1, finally obtain dry thallus 19.6g/L, fat content 33.5%.

Example 9

Referring to the literature (Sun Y, Cheng J J. Dilute acid pretreatment of rye straw andbermudagrass for ethanol production. Bioresour Technol, 2005, 96(14): 1599-1606), the rye straw was used as raw material, dried and crushed for 60 Mesh sieve, mix with 1.5% (w/w) dilute sulfuric acid, the solid-liquid ratio is 10% (w/w), digest at 121°C for 90min; filter, add cellulase (25FPU/g solid) and glucoside to the solid matter Enzyme (75IU/g solid), react at 50°C, 100rpm for 48h; filter, adjust the total reducing sugar concentration to 90g/L, including 56g/L glucose and 34g/L xylose. Add 1.2g/L yeast powder, 0.3g/L ammonium chloride, 0.9g/L magnesium chloride, 0.1g/L sodium sulfate, 6g/L potassium dihydrogen phosphate, 3g/L dipotassium hydrogen phosphate, Trace element solution 1% (v/v) was used to adjust the pH to 5.5. After sterilizing at 121°C for 15 minutes, the seed solution of Cryptococcus curvatus ATCC 20509 (purchased from the American Type Culture Collection) was inserted at 20% (v/v) inoculum. Incubate at 28°C for 132 hours with aeration. Fermentation was terminated, and the residual glucose and xylose concentrations in the fermentation broth were 3.1 g/L and 1.6 g/L respectively. Carry out post-treatment according to the method of Example 1, finally obtain dry thallus 23.8g/L, fat content 39.4%.

Compared with the conventional fermentation process in which glucose is preferentially utilized, the present invention relates to the synchronous utilization of glucose and xylose, which can eliminate the secondary growth phenomenon in the fermentation process, increase the substrate conversion rate, shorten the fermentation cycle, and thereby reduce production costs; and Compared with the method of biotransforming other raw materials to prepare microbial oil, the raw materials used in the present invention are lignocellulosic biomass containing glucose and xylose, such as trees, straw, etc., which have the advantages of abundant resources, wide sources, and low price, and the fermentation The method for obtaining oil is easy to implement and low in cost, and is a new way to produce microbial oil by utilizing renewable resources.

Claims (5)

1. the preparation method of a microbial oil is characterized in that: adopt the carbon source that contains glucose and wood sugar to cultivate oleaginous microorganism, obtain to contain the grease of one or more lipid acid and derivative thereof, glucose and wood sugar are by the oleaginous microorganism synchronous consumption;

Glucose content is 5%～95% in the described carbon source;

Described oleaginous microorganism is one or more among skin shape trichosporon Trichosporon cutaneum, crooked Cryptococcus Cryptococcus curvatus, the red winter spore Rhodosporidium toruloides.

2. according to the preparation method of the described microbial oil of claim 1, it is characterized in that:

Described oleaginous microorganism is cultivated and carry out 20 ℃～37 ℃ of culture temperature, pH4.0～8.0 under aerobic conditions.

3. according to the preparation method of the described microbial oil of claim 1, it is characterized in that: the described carbon source that contains glucose and wood sugar is that perhaps lignocellulosic material to be hydrolyzed to handle make by glucose and wood sugar is mixed.

4. according to the preparation method of the described microbial oil of claim 3, it is characterized in that: described lignocellulosic material is the biomaterial that contains Mierocrystalline cellulose, hemicellulose and xylogen.

5. according to the preparation method of the described microbial oil of claim 3, it is characterized in that: described lignocellulosic material is agricultural crop straw or forestry processing waste.