CN102851326A

CN102851326A - Method for preparing ethanol by simultaneous saccharification and fermentation of cellulose by using monilia tropicalis

Info

Publication number: CN102851326A
Application number: CN2012103045467A
Authority: CN
Inventors: 周雪琴
Original assignee: TAICANG ZHOUSHI CHEMICAL PRODUCT CO Ltd
Current assignee: TAICANG ZHOUSHI CHEMICAL PRODUCT CO Ltd
Priority date: 2012-08-24
Filing date: 2012-08-24
Publication date: 2013-01-02

Abstract

The invention provides a method for preparing ethanol by simultaneous saccharification and fermentation of cellulose by using monilia tropicalis. The present invention uses monilia tropicalis as a starting strain, and provides optimum process parameters for simultaneous saccharification and fermentation (SSF) of cellulose to produce ethanol.

Description

A kind of method of utilizing candida tropicalis simultaneous saccharification and fermentation Mierocrystalline cellulose to prepare ethanol

Technical field

The invention belongs to fermentation technical field, be specifically related to a kind of method of utilizing candida tropicalis simultaneous saccharification and fermentation Mierocrystalline cellulose to prepare ethanol.

Background technology

In chemical field, ethanol (being commonly called as alcohol) is a kind of important industrial raw material, be widely used in the fields such as chemical industry, food, drink industry, military project, daily-use chemical industry and medical and health, and ethanol can improve the fuel qualities mouth as a kind of good fuel (its fuel value reaches 26900 kJ/kg)].Nowadays, in fermentation industry, mainly adopt the yeast saccharomyces cerevisiae bio-transformation to produce ethanol.

Take biomass resource as raw material, the alcohol fuel of fermentative Production is of great significance for the Double jeopardy tool of alleviating current petroleum resources critical shortage and the deterioration of the ecological environment as renewable and clean energy resource.Yet in the existing ethanol fermentation production technology, the transformation efficiency of raw material and the yield of product are all lower, and high production cost has seriously restricted the alcohol fuel industrial expansion.Therefore, traditional ethanol fermentation technique is transformed, developing low-cost alcohol production new technology seems particularly urgent with the needs that adapt to the fuel ethanol industrial development.

SSF (simultaneous Sacchrification and Fermentation) method mainly is a kind of trial that improves enzymatic hydrolysis, be glucose once generation, namely be fermented into ethanol, constantly remove glucose, improve enzymatic hydrolysis speed, prevent that sugar accumulation and product from suppressing.Like this, decapacitation reduces outside the product inhibition, can also produce higher alcohol concn, eliminates expensive reaction and separation processes equipment, reduces cost, and reduction can not obtain the enzyme carrier requirement of single hydrolysis and fermentation.

Utilized the vegetable fibre fermentative production of ethanol after deliberation more than 20 year, the foreign scholar has carried out broad research to straw, bagasse, rice straw etc.The human dilute acid pretreatment straws such as Badal C., adopting the SSF method to obtain alcohol concn is 17.0 g/L.Human microwave and the Alkaline pretreatment rice straws such as Zhu Shengdong (transliteration), adopting the SSF method to obtain alcohol concn is 25.8 g/L.Kumari (U.S.) is plain with the Protoplast Fusion Strains M62 fermentation filter paper fibre of wooden mould QM9414 and yeast NCIM3288, and alcohol yied is 19.1g/L.Lark (Britain) has studied with waste paper (concentration is 190 g/L) and has produced ethanol, and the fermentation final concentration is 35 g/L.Domestic research to corn stalk is comparatively extensive, but is bacterial strain or mixed fungus fermentation after employing mutagenesis, the fusion mostly, and fermentation process adopts liquid fermentation method mostly.Zhang Jiquan etc. utilize Pichia ohmeri that the pretreated maize straw of dilute sulphuric acid is carried out liquid SSF fermentation, and alcohol yied is 0.327g/g.

Summary of the invention

The invention provides a kind of method of utilizing candida tropicalis simultaneous saccharification and fermentation Mierocrystalline cellulose to prepare ethanol.

1. method of utilizing candida tropicalis simultaneous saccharification and fermentation Mierocrystalline cellulose to prepare ethanol, when it is characterized in that fermenting, inoculum size by the certain volume mark accesses seed liquor in the fermention medium, adds simultaneously a certain amount of cellulase, ferments the regular hour at a certain temperature.

2. the preparation process of the described seed liquor of step 1: picking one ring bacterial classification from the good fresh inclined-plane of firm switching, access in the 50 mL seed culture mediums, place on the shaking table at 30 ℃, 160 r/min shaking culture 24 h.

3. the described fermention medium of step 1 (g/L): yeast extract paste 5, peptone 5, (NH ₄) ₂SO ₄2, KH ₂PO ₄L, MgSO ₄.7H ₂O 0.3.

4. the described seed culture medium of step 2 (g/L): glucose 20, yeast powder 10, peptone 20.

5. the described optimum fermentation temp of step 1 is 40 ℃.

6. the optimum amount of the described cellulase of step 1 is 25 U/g.

7. the described optimum inoculation amount of step 1 is 9%.

8. the described best fermentation time of step 1 is 72 h.

Beneficial effect of the present invention is: the present invention is take candida tropicalis as starting strain, by the optimal processing parameter of simultaneous saccharification and fermentation (SSF) cellulose raw producing and ethanol

Description of drawings

Fig. 1 leavening temperature is on the impact of SSF producing and ethanol.

Fig. 2 cellulase is on the impact of SSF producing and ethanol.

Fig. 3 inoculum size is on the impact of SSF producing and ethanol.

Fig. 4 fermentation time is on the impact of SSF producing and ethanol.

Embodiment

The following examples elaborate to the present invention, but to not restriction of the present invention.

The used bacterial strain of the present invention is candida tropicalis, buys in ACCC, is numbered: ACCC20004

Embodiment 1

The implementation case explanation leavening temperature is on the impact of SSF producing and ethanol, inoculum size by 6% (v/v) is inoculated in fermention medium, in the SSF fermention medium of enzyme dosage 15 U/g substrates, behind 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃, 42 ℃, 44 ℃, 46 ℃ lower 72 h of cultivation, measure respectively the weight of each Erlenmeyer flask, calculate CO ₂Weight loss.The result as shown in Figure 1.

As seen from Figure 1, along with the increase of leavening temperature, CO ₂Weight loss begins to be linear ascendant trend, and this is to help enzymolysis because temperature improves, but is unfavorable for the growth of yeast, so after temperature reaches 40 ℃, CO ₂Weight loss no longer rises and tends towards stability, and about 40 ℃ of temperature are that enzymolysis gets sugared and optimum temps ethanol conversion.

Embodiment 2

The implementation case explanation cellulase is on the impact of SSF producing and ethanol, inoculum size by 6% (v/v) is inoculated in fermention medium, enzyme dosage is respectively in the SSF fermention medium of 10 U/g, 15 U/g, 20 U/g, 25 U/g, 30 U/g, 35 U/g substrates, 40 ℃, cultivate the weight of measuring each Erlenmeyer flask behind 72 h, calculate CO ₂Weight loss, the result as shown in Figure 2.

As shown in Figure 2, enzyme dosage is between 10-15 U/g the time, CO ₂Weight loss is less; In the time of between 15-25 U/g, along with the increase of enzyme dosage, CO ₂Weight loss improves constantly; When enzyme dosage is 25 U/g, CO ₂Weight loss is maximum, illustrates that alcohol yied is higher at this moment; Enzyme dosage improves again, CO ₂Weight loss descends to some extent.May be because the increasing of enzyme dosage improves fast the content of reducing sugar, thereby suppressed the output of ethanol.Therefore, when enzyme dosage was the 25 U/g left and right sides, alcohol yied was higher.

Embodiment 3

The implementation case explanation inoculum size is on the impact of SSF producing and ethanol, inoculum size by 3%, 6%, 9%, 12%, 15%, 18%, 21% (v/v) is inoculated in fermention medium respectively, in the SSF fermention medium of enzyme dosage 25 U/g substrates, 40 ℃, cultivate the weight of measuring each Erlenmeyer flask behind 72 h, calculate CO ₂Weight loss, the result as shown in Figure 2.

As can be seen 3, inoculum size is when 3%-9%, along with the increase of inoculum size, CO ₂Weight loss also constantly increases, and when inoculum size is 9%, reaches maximum value, decreases subsequently, may be because the increase of inoculum size cause between the bacterial classification competition to nutrition, thus alcohol yied is reduced.

Embodiment 4

The implementation case explanation fermentation time is on the impact of SSF producing and ethanol, inoculum size by 9% (v/v) is inoculated in fermention medium, enzyme dosage is respectively in the SSF fermention medium of 25U/g substrate, 40 ℃, cultivating 24 h, 36h respectively, take out one bottle of weight of measuring Erlenmeyer flask behind 48 h, 60 h, 72 h, 84 h, calculate CO ₂Weight loss.The result as shown in Figure 4.

As seen from Figure 4, along with the prolongation of time, CO ₂Weight loss constantly increases, and reaches maximum during 72 h, tends towards stability subsequently, may be with the consumption of cellulase and bacterial strain aging relevant.Therefore, 72 h are the best fermentation time of SSF producing and ethanol.

Claims

2. method according to claim 1 is characterized in that, the preparation process of seed liquor: picking one ring bacterial classification from the good fresh inclined-plane of firm switching, access in the 50 mL seed culture mediums, and place on the shaking table at 30 ℃, 160 r/min shaking culture 24 h.

3. method according to claim 1 is characterized in that, fermention medium: yeast extract paste 5 g/L, peptone 5 g/L, (NH ₄) ₄SO ₄2 g/L, KH ₂PO ₄L g/L, MgSO ₄.7H ₂O 0.3 g/L.

4. method according to claim 2 is characterized in that, seed culture medium: glucose 20 g/L, yeast powder 10 g/L, peptone 20 g/L.

5. method according to claim 1 is characterized in that, leavening temperature is 40 ℃.

6. method according to claim 1 is characterized in that, the consumption of cellulase is 25 U/g.

7. method according to claim 3 is characterized in that, inoculum size is 9%.

8. method according to claim 1 is characterized in that, fermentation time is 72 h.