CN110894512A - Method for directly producing ethanol by using microalgae - Google Patents

Method for directly producing ethanol by using microalgae Download PDF

Info

Publication number
CN110894512A
CN110894512A CN201911337498.XA CN201911337498A CN110894512A CN 110894512 A CN110894512 A CN 110894512A CN 201911337498 A CN201911337498 A CN 201911337498A CN 110894512 A CN110894512 A CN 110894512A
Authority
CN
China
Prior art keywords
microalgae
fermentation
ethanol
gass
saccharomyces cerevisiae
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201911337498.XA
Other languages
Chinese (zh)
Inventor
贺诗欣
曲文颖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harbin Institute of Technology
Original Assignee
Harbin Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harbin Institute of Technology filed Critical Harbin Institute of Technology
Priority to CN201911337498.XA priority Critical patent/CN110894512A/en
Publication of CN110894512A publication Critical patent/CN110894512A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

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.

Description

Method for directly producing ethanol by using microalgae
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
Figure BDA0002331367350000031
Example 1. method for producing ethanol using microalgae.
The Saccharomyces cerevisiae used in this example was a mixture of Saccharomyces cerevisiae strains EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS at a ratio of 1: 1.
The microalgae is Chlamydomonas sp.QWY37, which contains COD,
Figure BDA0002331367350000032
And
Figure BDA0002331367350000033
has a carbohydrate productivity of 944 mg/L.d, and starch accounts for 58% by weight of the dry cells, and a method for producing ethanol is described in detail below.
1) Activating strains: inoculating the saccharomyces cerevisiae strain into a yeast leaching peptone glucose culture medium for culturing at the temperature of 30 ℃ and the stirring speed of 150rpm/min, after culturing for 72h, rinsing the obtained thalli after centrifugation for 2 times by using sterile water, and weighing;
2) fermentation culture: inoculating saccharomyces cerevisiae into a yeast extract peptone glucose culture medium containing microalgae, wherein the inoculation amounts of EG-D-CBHI-D-CBHII-D and BY-AASS/GASS/GASS are respectively 50g/L and 37 ℃, the stirring speed is 400rpm/min, and the inoculation amount of the microalgae in the fermentation culture medium is 300 g/L. The ethanol concentration was determined by gas chromatography (GC-2010, Shimadzu, Tokyo, Japan) according to the literature (Yamada et al, 2013), and the ethanol yield was determined to be 61g/L at QWY37, as shown in FIG. 1, after fermentation for 72 h.

Claims (7)

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.
CN201911337498.XA 2019-12-23 2019-12-23 Method for directly producing ethanol by using microalgae Pending CN110894512A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911337498.XA CN110894512A (en) 2019-12-23 2019-12-23 Method for directly producing ethanol by using microalgae

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911337498.XA CN110894512A (en) 2019-12-23 2019-12-23 Method for directly producing ethanol by using microalgae

Publications (1)

Publication Number Publication Date
CN110894512A true CN110894512A (en) 2020-03-20

Family

ID=69788104

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911337498.XA Pending CN110894512A (en) 2019-12-23 2019-12-23 Method for directly producing ethanol by using microalgae

Country Status (1)

Country Link
CN (1) CN110894512A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110982859A (en) * 2019-12-23 2020-04-10 哈尔滨工业大学 Method for producing carbohydrate by using pig-raising sewage

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5578472A (en) * 1993-09-27 1996-11-26 Mitsubishi Jukogyo Kabushiki Kaisha Process for the production of ethanol from microalgae
JPH10290698A (en) * 1997-04-18 1998-11-04 Mitsubishi Heavy Ind Ltd Production of ethanol using microalga
CN103103221A (en) * 2013-01-29 2013-05-15 广州分子生物技术有限公司 Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast
CN103421850A (en) * 2012-05-26 2013-12-04 北京大学深圳研究生院 Method used for producing bioethanol with Scenedesmusabundans
CN110982859A (en) * 2019-12-23 2020-04-10 哈尔滨工业大学 Method for producing carbohydrate by using pig-raising sewage

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5578472A (en) * 1993-09-27 1996-11-26 Mitsubishi Jukogyo Kabushiki Kaisha Process for the production of ethanol from microalgae
JPH10290698A (en) * 1997-04-18 1998-11-04 Mitsubishi Heavy Ind Ltd Production of ethanol using microalga
CN103421850A (en) * 2012-05-26 2013-12-04 北京大学深圳研究生院 Method used for producing bioethanol with Scenedesmusabundans
CN103103221A (en) * 2013-01-29 2013-05-15 广州分子生物技术有限公司 Method for converting cellulose into ethanol by use of mixed culture of genetically recombinant yeast
CN110982859A (en) * 2019-12-23 2020-04-10 哈尔滨工业大学 Method for producing carbohydrate by using pig-raising sewage

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
WENYING QU 等: ""Optimizing real swine wastewater treatment with maximum carbohydrate production by a newly isolated indigenous microalga Parachlorella kessleri QWY28"", 《BIORESOUR TECHNOL》 *
XIAOCHEN HUANG 等: ""Fermentation of pigment-extracted microalgal residue using yeast cell-surface display: direct high-density ethanol production with competitive life cycle impacts"", 《GREEN CHEMISTRY》 *
朱顺妮 等: ""微藻生物能源研究现状及展望"", 《新能源进展》 *
王鹏翔 等: ""微藻生物质生产燃料乙醇技术进展"", 《当代化工》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110982859A (en) * 2019-12-23 2020-04-10 哈尔滨工业大学 Method for producing carbohydrate by using pig-raising sewage
CN110982859B (en) * 2019-12-23 2023-05-05 哈尔滨工业大学 Method for producing carbohydrate by utilizing pig raising sewage

Similar Documents

Publication Publication Date Title
Chen et al. A comparative study between fungal pellet-and spore-assisted microalgae harvesting methods for algae bioflocculation
Ren et al. A new lipid-rich microalga Scenedesmus sp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production
WO2012012671A2 (en) Organism co-culture in the production of biofuels
CN105441524A (en) Method for increasing yield of microalgae grease with saccharose as carbon source through co-culture
US20080085536A1 (en) Production of Cellulose in Halophilic Photosynthetic Prokaryotes (Cyanobacteria)
CN109706089A (en) A kind of xylose-fermenting strains being resistant to mortifier and its construction method and application
CN103952312B (en) One strain limnetic chlorella Chlorella sorokiniana GS03 and application thereof
CN103555778A (en) Method for generating energy from hydrogen production bacteria and oily microalgae in stair-coupling mode
Alrubaie et al. Microalgae Chlorella vulgaris harvesting via co-pelletization with filamentous fungus
CN103421850A (en) Method used for producing bioethanol with Scenedesmusabundans
CN109456905B (en) Cryptococcus rhodochrous for promoting microalgae to utilize sucrose and application thereof
CN110894512A (en) Method for directly producing ethanol by using microalgae
CN110484571B (en) Method for semi-continuously producing hydrogen and grease by using corn straw
CN102226163B (en) Clostridium acetobutylicum strain and application thereof
Puangbut et al. Integrated cultivation technique for microbial lipid production by photosynthetic microalgae and locally oleaginous yeast
Suresh et al. Isolation and morphological identification of some indigenous microalgae from Ethiopia for phycoprospecting
CN103086582A (en) Methane preparation method
KR20110122424A (en) Novel fatty oilic microalgae krs101 and preparing method for biooil using thereof
Viswanath et al. Biodiesel production potential of wastewater microalgae Chlorella sp. under photoautotrophic and heterotrophic growth conditions
CN110885765B (en) Rhodotorula and culture method thereof and application of rhodotorula in production of biological grease
WO2019041567A1 (en) High-yield butanol clostridium and screening and use thereof
JP2012040006A (en) Method for producing oil by yeast of genus cystofilobasidium
CN107400673B (en) Synechocystis PCC6803 mutant strain and application thereof
KR101155435B1 (en) Novel strain Scenedesmus obliquus YSW15
CN103045491A (en) Mixed culture method of microorganism containing oil

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20200320