阅读说明：本技术 一种利用微藻直接生产乙醇的方法 (Method for directly producing ethanol by using microalgae ) 是由 贺诗欣 曲文颖 于 2019-12-23 设计创作，主要内容包括：一种利用微藻直接生产乙醇的方法,属于发酵工程技术领域。为了解决目前利用微藻生物合成乙醇的成本高,效率低的问题,本发明提供了一种利用微藻生产乙醇的方法,以表达淀粉酶和纤维素酶的酿酒酵母为发酵菌种,接种到含有微藻的发酵培养基中发酵培养制备乙醇；所述微藻为凯氏拟小球藻Parachlorella kessleri QWY28、栅藻Desmodesmus sp.QWY36或衣藻Chlamydomonas sp.QWY37。本发明克服了淀粉和纤维素水解的反应试剂毒性风险,降低了乙醇生产的成本,为真实养猪污水培养的富含碳水化合物的微藻直接生产高浓度乙醇工艺提供了新的视角。(A method for directly producing ethanol by using microalgae, belonging to the technical field of fermentation engineering. In order to solve the problems of high cost and low efficiency of ethanol biosynthesis by using microalgae at present, the invention provides a method for producing ethanol by using microalgae, wherein saccharomyces cerevisiae for expressing amylase and cellulase is taken as a fermentation strain and is inoculated into a fermentation medium containing the microalgae for fermentation culture to prepare the ethanol; the microalgae is Chlorella Kessleri QWY28, Scenedesmus sp.QWY36 or Chlamydomonas sp.QWY37. The invention overcomes the toxicity risk of reaction reagents for hydrolyzing starch and cellulose, reduces the cost for producing ethanol, and provides a new visual angle for the process for directly producing high-concentration ethanol by microalgae which is cultured by real pig-raising sewage and is rich in carbohydrate.)

1. A method for directly producing ethanol by using microalgae is characterized in that saccharomyces cerevisiae expressing amylase and cellulase is taken as a fermentation strain, and is inoculated into a fermentation culture medium containing the microalgae for fermentation culture to prepare the ethanol; the microalgae is Chlorella Kessleri QWY28, Scenedesmus sp.QWY36 or Chlamydomonas sp.QWY37, and is obtained by culturing and enriching the microalgae through the pig raising sewage, wherein the inoculation amount of the microalgae in the fermentation culture medium is 150-.

2. The method of claim 1, wherein the Saccharomyces cerevisiae is a mixture of EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS.

3. The method of claim 1, wherein the fermentation medium is a yeast extract peptone glucose medium.

4. The method according to claim 1, characterized in that the method is as follows:

1) activating strains: inoculating a saccharomyces cerevisiae strain expressing amylase and cellulase into a yeast leaching peptone glucose culture medium for culturing at the temperature of 20-30 ℃ and the stirring speed of 100-200rpm/min, culturing for 48-72h, and rinsing the obtained thalli after centrifugation by using sterile water and then weighing;

2) fermentation culture: then, the saccharomyces cerevisiae expressing amylase and cellulase is inoculated into a yeast extract peptone glucose medium containing microalgae, the inoculation amount is 50-100g/L, the stirring speed is 400rpm/min at 37 ℃, ethanol fermentation is carried out, and the inoculation amount of the microalgae in the fermentation medium is 300g/L according to the volume of the yeast extract peptone glucose medium.

5. The method of claim 4, wherein the centrifugation of step 1) is performed at 5200rpm/min for 10min at 4 ℃.

6. The method according to claim 4, wherein the Saccharomyces cerevisiae strain of step 2) is a mixture of EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS at a ratio of 1:1, and the inoculation amount of EG-D-CBHI-D-CBHII-D is 50g/L and the inoculation amount of BY-AASS/GASS/GASS is 50g/L based on the volume of yeast extract peptone glucose medium.

7. The method according to claim 4, wherein the method for collecting ethanol after fermentation in step 2) comprises the following steps: adding fermented broth into round-bottom flask of distillation apparatus, distilling in water bath at 85-95 deg.C, and collecting effluent ethanol.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for directly producing ethanol by using microalgae.

Background

The photosynthetic organisms utilize light energy to fix CO₂The alga refers to a photosynthetic organism having high photosynthetic efficiency under good culture conditions. The industrial culture of algae has been carried out for more than half a century, and people use algae as industrial raw material, fuel and feedThe demand for raw materials for materials, fine chemicals and health products is increasing, and algae is considered to be an important place in the future industry. Due to CO₂Various useful carbon components are produced during the fixing process, and thus various carbon components produced by algae become a research hotspot during the cultivation of algae.

Due to concerns about exhaustion of fossil fuels, there is a growing need for early search of alternative fuels, and due to increased health consumers, there is an increased demand for functional chemicals more suitable for maintaining and improving health, however, the shortage of fresh water makes the actual production cost of microalgae bioethanol too high, and therefore, there is a need for development of a low-cost, high-carbohydrate production culture system to better meet the actual demand.

Disclosure of Invention

In order to solve the problems of high cost and low efficiency of ethanol biosynthesis by using microalgae at present, the invention provides a method for producing ethanol by using microalgae, in particular to a method for producing ethanol by using microalgae, wherein saccharomyces cerevisiae expressing amylase and cellulase is used as a fermentation strain and is inoculated into a fermentation culture medium containing the microalgae for fermentation culture to prepare the ethanol; the microalgae is Chlorella Kessleri QWY28, Scenedesmus sp.QWY36 or Chlamydomonas sp.QWY37, and is obtained by culturing and enriching the microalgae through the pig raising sewage, wherein the inoculation amount of the microalgae in the fermentation culture medium is 150-.

Further, the Saccharomyces cerevisiae is a mixed strain consisting of EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS.

Further defined, the fermentation medium is a yeast extract peptone glucose medium.

Further defined, the method for producing ethanol using microalgae is as follows:

1) activating strains: inoculating a saccharomyces cerevisiae strain expressing amylase and cellulase into a yeast leaching peptone glucose culture medium for culturing at the temperature of 20-30 ℃ and the stirring speed of 100-200rpm/min, culturing for 48-72h, and rinsing the obtained thalli after centrifugation by using sterile water and then weighing;

2) fermentation culture: then, the saccharomyces cerevisiae expressing amylase and cellulase is inoculated into a yeast extract peptone glucose medium containing microalgae, the inoculation amount is 50-100g/L, the stirring speed is 400rpm/min at 37 ℃, ethanol fermentation is carried out, and the inoculation amount of the microalgae in the fermentation medium is 300g/L according to the volume of the yeast extract peptone glucose medium.

Further defined, the centrifugation described in step 1) centrifuged the cells at 5200rpm/min for 10min at 4 ℃.

Further limiting, the saccharomyces cerevisiae strain in the step 2) is a mixed strain composed of EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS according to a ratio of 1:1, and the inoculation amount of EG-D-CBHI-D-CBHII-D is 50g/L and the inoculation amount of BY-AASS/GASS/GASS is 50g/L based on the volume of a yeast extract peptone glucose medium.

Further limiting, the method for collecting the ethanol after fermentation in the step 2) comprises the following steps: adding fermented broth into round-bottom flask of distillation apparatus, distilling in water bath at 85-95 deg.C, and collecting effluent ethanol.

Advantageous effects

Before fermenting microalgae by using yeast which is not genetically modified, the microalgae needs to be hydrolyzed to remove structures such as cellulose which affects fermentation in the microalgae, and the invention utilizes the microalgae which is obtained by screening and culturing the pig-raising sewage to directly produce high-concentration ethanol, and the ethanol is produced by fermentation within 72 hours under the fermentation of saccharomyces cerevisiae, wherein the yield can reach 61g/L, the toxicity risk of a reaction reagent for hydrolyzing starch and cellulose is overcome, the ethanol production cost is reduced, the invention is a novel and economic technology, and provides a new visual angle for the process for directly producing the high-concentration ethanol by using the microalgae which is cultured by using the real pig-raising sewage and is rich in carbohydrate.

Drawings

FIG. 1 shows the fermentation principle of Saccharomyces cerevisiae expressing amylase and cellulase, the starch utilization and the time course of direct ethanol production.

Detailed Description

The saccharomyces cerevisiae used in the invention: EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS are described in Huang, X, Bai, S, Liu, Z, Hasunuma, T, Kondo, A., & Ho, S.H. (2019), Fermentation of fragment-extracted microbial depletion using yeast cell-surface display: direct-dense ethylene product with comparative chemistry experiments.https:// doi.org/10.1039/C9GC 02634G. The strain can be obtained from Harbin university of industry.

In order to better understand the principle of ethanol production by using microalgae as a substrate on a culture medium containing microalgae by using recombinant yeast, the invention provides the gene composition and metabolic route of corresponding yeast, which is shown in fig. 1, wherein the genes and the related plasmids mentioned in the figure are also recorded in the text and supplementary materials of the article, and are not repeated herein.

The microalgae of the invention: the Chlorella Kessleri QWY28, Scenedesmus sp.QWY36 or Chlamydomonas sp.QWY37 is obtained by separating cells from a river near a pig farm in Harbin, China at 30 deg.C and 200. mu. mol/m²S. under light conditions for 24h, using BG-11 solid medium, the algal strains were subcultured every 2 weeks, identified by morphological observation and 18S rDNA sequence comparison, etc., as Chlorella vulgaris Parachlorella kesleri QWY28, Scenedesmus Desmodus sp.QWY36 or Chlamydomonas sp.QWY37 (shown in FIG. 1), the 18SrDNA sequences of algal strains QWY28, QWY36 and QWY37 were submitted to the American national center for Biotechnology information, having serial numbers MK367466, MK367467 and MK367468, respectively, in the case of Chlamydomonas, the visible cells are oval in size about 10 μm, the cells have 2 hairs in size about the same as the length of the cells, the cells have growth by binary fission, physiological or biochemical characteristics ①: growth in real pig photosynthetic performance: use of flagellar for autotrophic growth, the chlorophyll growth by use of flagellar, and the pH range 357: 35. the photosynthetic growth range of the algal strains, 35. the photosynthetic substance, the pH 7: 0.5635. the growth range of Chlamydomonas the photosynthetic substance, the pH 0.5630. the growth range, 35. the growth by use of Chlamydomonas the photosynthetic substance.

The above fungi and algae are described in: qu, W., Zhang, C, Zhang, Y, Ho, S, 2019.Optimizing realswine water turbine with maximum carbohydrate production by a new isolatedoriented microalalia bessorella kesleri QWY28. biorsesourceHNECHNOLOGY 289. the public is available through Harbin university of Industrial science.

The BG-11 medium formulation is shown in Table 1:

TABLE 1 BG-11 Medium formulation