Study on conversion of fuel ethanol by straw cellulose fermentation

abstract

China is rich in straw resources, with an annual output of over 300 million tons. Producing fuel ethanol from straw, right?

China has great potential and broad application prospects in energy issues, grain saving instead of grain and environmental protection in the future. rice straw

The main component is cellulose, and the most important limiting factor of cellulose utilization is to convert cellulose into fermentable cellulose.

There are two main ways to solve this problem: (l) improving the economy of cellulase production, mainly involving high cellulase content.

Obtain cellulase production technology, improve its synthesis efficiency to reduce the unit cellulase production cost; 2. Mention

The utilization efficiency of protease mainly involves the hydrolysis and catalysis of cellulase to reduce the production cost of fermentable reducing sugar per unit.

In this study, the characteristics of enzyme production were studied from the perspective of strain breeding, and the fermentation medium was optimized by response surface methodology.

The technology of producing high-activity cellulase by batch fermentation in SL fermentor was introduced. Isolation and purification of cellulase. Cultivate a generation

Engineering strain of saccharomyces cerevisiae; The immobilized fermentation of Saccharomyces cerevisiae engineering bacteria cells was studied, and the two-stage fermentation method was adopted

Bioreactor disaster system cooperates with enzymatic hydrolysis of straw to produce fuel ethanol. The main results are as follows

1. A strain with high cellulase production (Penicillium YT0 1) was screened out. Protoplasts were induced by ultraviolet rays.

Mutants YT02 and YT02 use rice straw as carbon source, bean cake powder and sulfuric acid money as nitrogen source, and at 29℃, the initial P

Fermentation 12Oh, cellulase activity reached the highest, filter paper enzyme activity (FPA) and CMC enzyme activity (CMcas) were fermented in shake flask.

Glucose oxidase activity (CB) reached 3.86IU/mL, 207.4 1IU/mL and 1.4 iu/mL, respectively.

2. The composition of fermentation medium optimized by response surface method is: straw 4 1.95 L,

It's 24.83g/L, the number of skins is 22. 16ba, it's H4)2504, KHZpO4 is 4g/L, MgSO because of 0.sg/L;. Originated from

After 120h fermentation in the optimized medium, the filter paper enzyme activity, cMc enzyme activity and β -glucosidase activity reached the highest respectively.

The international unit/ml, 357.4 1IU/ ml and 3.704 IU/ ml are much higher than the cellulase activity level before optimization.

3. The effects of temperature, pH value and dissolved oxygen on cell growth and enzyme production were studied in SL fermentor.

The technological conditions of fermentation are as follows: fermentation temperature is ℃, 0-32h, and dissolved oxygen is 70%; Adding H to 1 will lead to fermentation.

29oc, dissolved oxygen 50%, initial pH value of fermentation broth 6.0, filter paper enzyme activity, CMC enzyme activity and P- glucose after fermentation for% h..

The enzyme activities reached11.13 iu/ml, 465.24 iu/mL and 4.08IU/mL, respectively, which were higher than those of shake flask fermentation.

The fermentation kinetics showed that the growth of mutant YT02 was partly related to cellulase components.

4. Two kinds of endonucleases were isolated and purified by decapitation A-25 and sephadexG-75.

(CMCase) and a p- glucoamylase, the purification multiple of CMCase is 13.48, and the recovery rate is 10.54%.

The purification multiple of glycosidase was 18.62, and the recovery rate was 8.62%. SDS-PAGE obtained single protein molecular bands.

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The contents were 73kDa, 43kDa and 57.8kDa, respectively, and were analyzed by n-terminal sequencing and mass spectrometry.

5. The recipient strain of this engineering strain is Saccharomyces cerevisiae NAN-27, which has excellent ethanol production performance. use

PYMIKP is a good multi-copy integration vector, which can integrate cellobiose metabolism gene BGLI into Saccharomyces cerevisiae.

Physically. Therefore, a stable cellobiose metabolic pathway was established in the industrial strains of Saccharomyces cerevisiae, thus expanding the alcohol production.

The utilization range of cellulose reduces the inhibitory effect of cellobiose on cellulase hydrolysis. Calcium alginate gel was used for embedding and fixation.

Compared with free cells, the immobilized cells of cellobiose Saccharomyces cerevisiae have shorter fermentation time and higher ethanol yield.

Can effectively utilize the rice straw hydrolysate for alcohol fermentation.

6. The substrate, enzymolysis temperature, enzymolysis pH, substrate concentration and the properties of cellulase were studied.

And other key factors are studied. Due to the low activity of cellulose dicarboxylate in YT02 cellulase system (CB/F team is

The ethanol conversion rate of straw cellulose pretreated with dilute acid and alkali is only 65438 08%. Use metabolic cellobiose

The inhibitory effect of cellobiose on enzymatic hydrolysis can be partially relieved by free cell fermentation of parent engineering bacteria, and rice straw cellulose can inhibit B.

The rate can be increased to 20%. In addition, the immobilized cellobiose metabolizing Saccharomyces cerevisiae workers were embedded in calcium alginate gel.

The conversion rate of fermented ethanol and straw cellulose can reach 26%. The research results in this field are helpful to understand fibers in depth.

Co-degradation mechanism.

7. Enzymatic hydrolysis and immobilization of fiber raw materials are organically related to the function of Saccharomyces cerevisiae engineering bacteria.

The new two-stage series bioreactor can effectively remove fiber 2 under the synergistic effect of the reactor system.

The feedback inhibition of glucose on cellulase promoted the enzymatic hydrolysis of rice straw, and the fermentation lasted for 40h, B.

Up to 25.5 L, the conversion rate of cellulose to ethanol reached 43.0% (the theoretical conversion rate of cellulose to ethanol was 56.438+0

It is 1.65 times that of SSF, and the production efficiency is 0.64 Lh. Batch feeding

Enzymatic fermentation technology can increase the final concentration of fiber substrate to 250 years old, and the final concentration of ethanol product to 66.5 1, leaving l,

The utilization rate of cellulase and the production efficiency of ethanol are improved, and the production cost of ethanol is reduced. The reactor has stable performance.

High efficiency, reusable immobilized cells and convenient automatic control.

Key words: cellulase, rice straw, Saccharomyces cerevisiae, fuel ethanol, series bioreactor.

catalogue

abstract .................................................................

Abstract .......................................................... ... ……IH

The first chapter is literature review.

Rice straw resources and its degradation mode ...............................................

1. 1 composition and structure of straw ..., ..................................., ... l

1.2 Pretreatment of ..................................................... with Rice Straw 3

1.2. 1 physical pretreatment of straw ............................................. 3

1.2.2 Chemical pretreatment of rice straw ............................................. 3

1.2.3 Biological Pretreatment of Rice Straw ............................................. IV

1.3 degradation model of straw cellulose ............................................. iv

1.3 1 acid hydrolyzed rice straw ................................................... 5

1.3.2 Enzymatic hydrolysis of ................................................ from straw ...

2 the nature and use of cellulase ................................................ 6

2. 1 multi-enzyme system ................................................ cellulase 6

2.2 Molecular Structure of Cellulase ................................................ VII

2.3 Mechanism of Action of Cellulase ................................................... 9

2.4 molecular weight of cellulase ................................................ 10

2.5 Optimum reaction conditions and stability of cellulase ...................................... 1 1

2.6 the application of cellulase ...................................................... H.

3 Cellulase production ........................................................ 12

3. 1 Breeding of Cellulase Producing Strain .............................................. 12

3.2 Production of Cellulase ...................................................... 14

4 Bioconversion of Rice Straw into Fuel Ethanol .......................................... 15

4. 1 Advantages and application status of fuel ethanol .......................................... 15

4.2 Method of Bioconversion of Fuel Ethanol from Straw Cellulose ............................. 16

4.2. 1 ........................................ for producing fuel ethanol by stepwise hydrolysis and fermentation 16

4.2.2 Fuel ethanol production in ..................................... through simultaneous saccharification and fermentation ... 16

I

4.2.3 Fuel ethanol production ..................................... is fermented by immobilized cells ... 17.

4.3 The yeast pathway project of Youliang Liquor was applied to the production of fuel ethanol ................................ 17.

5. The purpose, significance and main contents of this study .......................................... 19.

5. 1 purpose and significance of this study .................................................. 19

5.2 The train of thought and technical route of this study .............................................. 20

5.3 The main content of this study is .................................................... 2 1

Chapter II Breeding of Cellulase-producing Bacteria and Study on Enzyme-producing Conditions ............................ 23

1 materials and methods ............................................................. 23

1. 1 material ................................................................ 23

1. 1. 1 reagent and solution preparation ....................................................

1. 1.2 strain and strain isolation source .................................................. 24

1. 1.3 medium ............................................................ 24

1. 1.4 main instruments and equipment .................................................... 25

1.2 method ................................................................ 25

Pretreatment of straw .............................., ..., .............. 25

1.2.2 Isolation and purification of cellulase-producing bacteria ..................................... 25

1.2.3 Preliminary identification of cellulase-producing bacteria .......................................... 25

1.2.4 Mutation ............................... Mutated Protoplast of Cellulase-producing Strain by Ultraviolet ... 25

A 1.2.5YTOZ liquid fermentation culture method for producing cellulase ............................... 26.

1.2.6 Enzymatic Hydrolysis of ..................................... from Different Pretreated Straws ...

1.2.7 analysis method .......................................................... 27

2 Results and Analysis ............................................................ 29

2. 1 Contents of components in different pretreated rice straw ................................... 29

2.2 Isolation and Screening of Cellulase-producing Bacteria ....................................... 29

2.3 identification of cellulose-producing strain YT0 1 ......................................... 3 1

2.4 The protoplast of cellulase-producing strain YT0 1 was induced by ultraviolet in ............................ ... 3 1

2.5 Effect of liquid fermentation medium composition and fermentation conditions on cellulase production of YT02 ................. 32

2.5. 1 Effects of different carbon sources on the enzyme production of YT02 ......................................... 32

2.5.2 Effects of Different Pretreatment of Rice Straw on Enzymatic Production of YT02 ............................... 33

2.5.3 Effects of Different Nitrogen Sources on Cellulase Production of YT02 ................................... 34

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2.5.4 Effect of adding microcrystalline cellulose on the cellulase production of YT02 ........................... 35

2.5.5 Effects of Different Inorganic Salts on YT0 1 Cellulase Production ................................. 35

2.5.6 effect of initial pH on YT0 1 cellulase production .................................... 36

2.5.7 Effect of Liquid Load on Cellulase Production of YT02 ..................................... 37

2.5.8 Effect of rotating speed on cellulase production of YT02 ....................................... 37

2.5.9 Effect of culture temperature on cellulase production of YT02 ................................... 38

2.5. Effect of inoculation amount of10 on cellulase production of YT02 .................................... 39

2.5.n Effect of culture time on the enzyme production of YT02 ........................................ 40

2.6 Study on Enzymatic Properties of Cellulase .............................................. 4 1

2.6. Effect of1temperature on the activity of cellulase components ................................ ................................................

2.6. the influence of zph on the activity of cellulase components ..................................... 4 1

2.7 Enzymatic hydrolysis of different pretreated rice straw by cellulase .............................. 42

3 Conclusion and discussion ............................................... 4: L.

3. 1 About the screened high-yield cellulase strain ....................................... 4: 3.

3.2 Transformation of Cellulase-producing Bacteria ...............................................

3.3 Penicillium YT02 enzyme production conditions and enzymatic characteristics of ........................................ 44

Chapter III Optimization of Fermentation Medium for Cellulase Production by YT02 ............................. 45

1 materials and methods .................................... 45

1. 1 material ................................................................ 45

1. 1. 1 reagent ................................................ 45

1. 1.2 test strain ....................................... 45

1. 1.3 medium ................................................ 45

1. 1.4 main instruments and equipment .................................................... 46

1.2 method ................................................................ 4.

1.2. 1 experimental design ............................................. 46

1.2.2 cultivation method ............................................. 46

1.2.3 analysis method .......................................................... 46

2 Results and Analysis .............................................. 47

2. 1 partial factor experiment to screen the main influencing factors of fermentation medium ............................ 47.

2.2 The steepest climbing experiment is close to the best point of fermentation medium, .................................... 50.

1 1 1

2.3 Optimization of YT02 Fermentation Medium Composition ................................. 5 1

2.4 Determination results of PH, residual reducing sugar and cellulase changes during fermentation .................... 59

3 Conclusion and discussion ..................................., ..............., 6 1

Chapter 4 Study on the Production of Cellulase by Batch Fermentation of YT02 ................................... 63

Materials and Methods ............................................................ 63

. 1 material ................................................................ 63

. 1. 1 reagent .............................................................. 63

. 1.2 strain .............................................................. 63

. 1.3 medium ............................................................

. 1.4 main instrument .......................................................... 64

way ........................

. 1 Seed culture for batch fermentation ...........

. …64

. …64

1.2.2 Effect of batch fermentation at constant temperature on cellulase production of YT02 ................................ 64

1.2.3 Effect of batch fermentation at variable temperature on cellulase production of YT02 ................................ 64

1.2.4 Effect of dissolved oxygen on the production of cellulase by batch fermentation of YT02 .............................. 64

1.2.5 effect of dissolved oxygen on batch fermentation of YT02 to produce cellulase ............................ 65

1.2.6 analysis method .......................................................... 65

2 Results and Analysis ............................................................ 65

2. 1 Effect of fermentation temperature on the yield of cellulase produced by YT02 ................................. 65

2.2 Effect of variable temperature fermentation on the cellulase production of YT02 ................................. 68

2.3 the effect of dissolved oxygen on the production of cellulase by YT02 ..................................... 69

2.4 The effect of staged dissolved oxygen batch fermentation on the cellulase production of YT02 ......................... 72

3 Conclusion and discussion ............................................................ 73

The fifth chapter is the separation, purification and enzymatic properties of cellulase produced by YT02. ..............

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Materials and methods …

. 1 material ...

. 1. 1 test material.

. 1.2 main reagent.

....……76

.....……76

.3 Common stock solution and buffer solution .......................................

1. 1.4 main instrument ........................................................... 78

1.2 method ................................................................. 78

1.2. 1 protein concentration determination method .............................................. 78

1.2.2 Isolation and purification of cellulase ............................................. 79

1.2.3 the cellulase was purified by SDS-PAGE gel electrophoresis and its relative molecular weight .............. 83 was determined.

N-terminal sequencing of 1.2.4 enzyme protein .................................................. 85

Mass spectrometry analysis of 1.2.5 enzyme protein .................................................. 86

2 Results and Analysis ............................................................ 87

2. 1DEAE-SephadexA-25 anion exchange chromatography results ................................

2. The relationship between1.1tubulin and NaCI concentration in elution buffer is ........................ 87.

2. Detection of tubular enzyme protein activity collected by1.2 chromatography ..........................................

2.25 is the result of hadexG-75 molecular sieve gel filtration chromatography, and ................................. is 88.

2.2. 1SephadexG-75 molecular sieve gel filtration chromatography to separate enzyme protein. .........................

2.2.2 Determination of cellulase activity by molecular sieve gel filtration chromatography ........................... 88

2.3 Purification of Cellulase .......................................... 89 in different purification steps

2.4SDS-PAGE polyacrylamide gel electrophoresis .......................................... 90

2.4. 1SDS-PAGE silver staining results ................................

2.4.2 results of molecular weight determination of cellulase by SDS-page gel electrophoresis ........................... 9 1

2.5 N-terminal sequencing results of enzyme protein ................................................ 9 1

2.6 ................................................ 93 results of mass spectrometry analysis of enzyme protein

3 Conclusion and discussion ............................................................ 94

Chapter VI Study on the Construction of Cellulose Metabolism Pathway and Cell Immobilization in Saccharomyces cerevisiae ................ 96

Materials and methods ........................................................................................

1 material ................................................................ 96

1. 1 strain and plasmid ........................................................ 96

1.2 ............................................. An enzyme and reagent for molecular cloning 96

1.3 preparation of rice straw hydrolysate .............................................. 97

Two methods ................................................................ 98

2. 1 Construction method of recombinant plasmid pYMIKP containing cellobiase gene (and curse 1).

2.2 Construction of cellobiose metabolic pathway in Saccharomyces cerevisiae ................................ 99

1.2.3 immobilization method of Saccharomyces cerevisiae engineering bacteria cells ................................... 10 1

1.2.4 fermentation method of immobilized yeast cells ........................................... 102

1.2.5 analysis method ......................................................... 102

2 Results and Analysis ........................................................... 103

2. 1 Results of expression and construction of recombinant strain with regulated gene .................................. 103.

2. 1. 1 target gene and obtained .............................................. 103.

2. 1.2 construction of recombinant plasmid containing target gene .................................... 103

2. 1.3 obtained the transformant of Saccharomyces cerevisiae NAN-27, ........................... 104.

2. 1.4 .......................... 1 () 4.

2.2 the influence of different immobilization conditions on the immobilization of NAN-28 cells results ........................ 105.

2.2. 1 determination results of different solvents for deoiling the transformed fibers of immobilized cells .................................................................................................................................. 65438+ 15.

2.2.2 Effects of different concentrations of sodium alginate on gel properties of immobilized cells .................... L () 5

2.2.3 Influence of yeast embedding amount on the result of cellobiose transformation by immobilized cells .....................! 06

2.3 Experimental results of batch fermentation of immobilized cells and free cells ............................... l () 6

2.4 results of repeated batch fermentation of immobilized cells ..................................... 107

3 Conclusion and discussion ........................................................... 108

3. 1 Construction of cellobiose metabolic pathway in Youliangjiu yeast ....................................... 108

3.2 Immobilization of Engineering Saccharomyces cerevisiae Cells .......................................... 1 10

Chapter VII Study on Production of Fuel Ethanol by Tandem Bioreactors ................. 1 12

Materials and methods ........................................................... 1 12.

L material .......................................................... 1 12.

1. 1 reagent ............................................................ 1 12.

1.2 strain ............................................................ 1 12

1.3 main instruments and equipment .................................................. 1 12

Methods .............................................................. 1 12.

2. 1 Pretreatment of Rice Straw Powder ................................................... 1 12

2.2 preparation of cellulase ................................................ 1 13

2.3 enzymatic saccharification of rice straw powder ................................................. 1 13

2.4 ......................................... 1 14 of bio-conversion of rice straw into fuel ethanol

2.5 determination method ......................................................... 1 15.

Visual recognition system

Results and analysis ........................................................... 1 16

2. 1 Effects of different pretreatment methods on the saccharification of rice straw Results ............................ 1 16.

2.2 Effects of different temperatures on the saccharification effect of rice straw Results ............................... 1 16.

2.3 the effect of different pH values on the saccharification of rice straw powder results .................................. 1 17.

2.4 Effect of different enzyme dosage on saccharification of rice straw powder Results ............................... 1 18.

2.5 Effects of different substrate concentrations on the saccharification effect of rice straw powder Results ............................. 1 18.

2.6 the results of SSF of rice straw .................................... 1 19.

2.7 ................................... 120 for ethanol production from rice straw in series reactors.

2.7. 1 ......................... 120.

2.7.2 Stability results of series bioreactor ..................................... 12 1.

2.7.3 ...........................122, results of fuel ethanol production by fed-batch and enzyme fermentation.

3 Conclusion and discussion ...................................................... 122

3. 1 ethanol production by two-stage bioreactor in series ....................................... 122

3.2 ......................................... 123: fed-batch collaborative enzymolysis fermentation process

3.3 Make full use of straw resources ............................................... 123

Chapter 8 Conclusion ........................................................ 124

Mainly refer to ......................................................... 126.

List of English abbreviations and main symbols .................................................. 146

Characteristics and innovation of this study .................................................... 147

Published and to be published academic papers and achievements .......................................... 148

Due to ............................................................... 149

Author's brief introduction ............................................................. 150

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