WO2010083652A1

WO2010083652A1 - Production of organic solvent by different combination of wastes

Info

Publication number: WO2010083652A1
Application number: PCT/CN2009/070288
Authority: WO
Inventors: Ting Hong-Hoi; Sharon Xiaorong Wang; Wenhua Huang
Original assignee: Breeze Global, Inc.
Priority date: 2009-01-23
Filing date: 2009-01-23
Publication date: 2010-07-29

Abstract

The residue from anti-biotic fermentation is used as the N resource in the Clostridium fermentation. A method of Clostridium fermentation with substrate containing the residue from anti-biotic fermentation as N resource, and a culture medium containing the residue from anti-biotic fermentation for Clostridium fermentation are provided.

Description

PRODUCTION OF ORGANIC SOLVENT BY DIFFERENT COMBINATION OF WASTES

Technical field

This invention relates to the use of bio-wastes in the fermentation, and, in particular, to the production of Bio-fuel by using bio-wastes as substrates and additives through Clostridium fermentation.

Technical Background

During the late 1970s and early 1980s, there was considerable international interest among various governments and scientific communities to review the production of alcohol fuels from easily and widely-produced renewable resources. A mixed degree of success was achieved in various countries with the bioconversion of alcohol fuel. This resulted in partially depressed crude oil market price which made it clear that fuel alcohol process development research should now be directed toward resolving those problems associated with the efficiency of both new and traditional processes.

Butanol is largely compatible with, and in some ways better than, gasoline. Its air/fuel mixture (Stoichometric A/F ratio) is 11.2 (Standard Gasoline is 14.7, and ethanol is 9.) which allows butanol to function in a standard gasoline engine. Its energy content is about 105,000 Btu per US gallon (Standard Gasoline has about 114,000 Btu per US gallon). In effect, butanol has about 92% of the energy of gasoline. In actual driving conditions, as butanol has a strong power and torque content, drivers will use a lighter foot on the accelerator and hold a higher gear longer, fuel efficiency will approximately match that of gasoline. It can be mixed with gasoline in any ratio in unmodified engines. Additionally, as butanol has a very low vapor pressure point (RVP 0.3) and a high Flash Point (FP 37 degrees Celsius), it is a very safe fuel to use in high temperatures. Butanol can be produced at an estimated cost of 85 cents per gallon, and is a direct replacement for gasoline, which ethanol cannot be. Butanol also has a high octane number (CN25, diesel averages CN45, ethanol CN9) which allows butanol to be blended with petro diesel and with vegetable oils (where it also reduces the gel temperature point and the viscosity) to produce biodiesel, with some positive environmental effects. Consequently, butanol is a very versatile fuel and fuel extender in both gasoline and diesel engines. It can do things that ethanol will never be able to do. Its manufacture from biomass will enhance the progress towards a bio-fuel world.

Vast majority of manufacturers still use corn wet milling process in the plant. Corn is soaked, followed by grinding, fine grinding, sieving, and centrifugation. After grinding, the germ containing oil is removed by floatation. Sieving results in the removal of fiber while centrifugation results in the separation of gluten. Then, batch fermentation is used for bioconversion of starch to butanol. But the cost of corn grows rapidly, leading to the results that the cost of raw materials is more than 70 % of the total cost of fuel production. Further, there are various wastes in different fields such as food wastes, the Cellulus after Anti-biotic fermentation (or CAB), and Brewery wastes. However, the utilization or the treatment of the wastes is not fully satisfactory.

Therefore, it is an urgent need to develop some inexpensive substrates at a large volume which can substitute grains and their derivatives for bio-fuel production. Further, it is an urgent need to develop a low cost and efficient process for producing bio-fuel.

Summary of Invention

One purpose of the invention is to provide an inexpensive substrate derived from bio-wastes for bio-fuel production. Another purpose is to provide a new method to bio-fuels such as butanol, ethanol, and acetone.

In the first aspect, the invention provides a use of the residues from anti-biotic fermentation or CAB as the N resource in the Clostridium fermentation. In the second aspect, the invention provides a culture medium useful for Clostridium fermentation wherein said culture medium contains the residues from anti-biotic fermentation or CAB as N resource.

In a preferred embodiment, said culture medium further contains a C resource or carbon source selected from the group consisting of Malt wine Distiller's spent grains, Palm oil mill effluent (POME), Food waste (FW), Corn stem stock (CSS), Wheat and Grain straw (WGS), Brewery waste (BW), sugar, starch, and the combinations thereof.

In a preferred embodiment, the ratio of said CAB and said C resource by dry weight is from 100:50 to 100:500, preferably 100: 100 to 100:300.

In the third aspect, the invention provides a method of Clostridium fermentation comprising the following step(s):

(a) Culturing the Clostridium bacteria under a fermentation condition suitable for Clostridium growth, wherein said fermentation condition includes the presence of residues from anti-biotic fermentation or CAB as N resource for Clostridium fermentation.

In a preferred embodiment, the said fermentation condition further includes the presence of a C resource selected from the group consisting of Malt wine Distiller's spent grains, Palm oil mill effluent (POME), Food waste (FW), Corn stem stock (CSS), Wheat and Grain straw (WGS), Brewery waste (BW), sugar, starch, and the combinations thereof.

In a preferred embodiment, the ratio of said CAB and said C resource by dry weight is from 100:50 to 100:500.

In a preferred embodiment, the method further comprises Step (b):

(b) separating or isolating one or more products from fermentation broth selected from the group consisting of: acetone, ethanol, butanol, Isopropanol, and hydrogen.

In a preferred embodiment, said CAB is prepared by the following steps: centrifuging the anti-biotic fermentation broth, thereby obtaining bacterial precipitate; treating said bacterial precipitate with 0.01-lN HNO₃, and adjusting pH to 4.0-8.0.

In the fourth aspect, the invention provides a method for producing bio-fuel by Clostridium fermentation comprising the following step(s):

(i) in the presence of residues from anti-biotic fermentation or CAB; and (ii) separating or isolating one or more products from fermentation broth selected from the group consisting of: acetone, ethanol, butanol, Isopropanol, and hydrogen.

In a preferred embodiment, the method further comprises: separating H₂ from fermentation broth.

In a preferred embodiment, the fermentation is solvent fermentation. In the fifth aspect, the invention provides a use of the residues from anti-biotic fermentation or CAB in the preparation of culture medium.

In a preferred embodiment, the culture medium is used for Clostridium fermentation.

DETAILED DESCRIPTION OF THE INVENTION After intensive and extensive study, the inventors have unexpectedly found that the Cellulus after Anti-biotic fermentation (or CAB) can be used as an inexpensive and ideal N resource or substrate in the fermentation. In particular, CAB is rich in proteins, free amino acids, and vitamins. Although these ingredients contribute nothing as substrate for saccharide-butanol bioconversion, they are helpful to the fast growth of the cell line in the culture medium, thereby shortening the fermentation time dramatically. The inventors completed the present invention based on the above.

Further, based on our longtime, broad yet in depth research on Food waste (FW) and Cellulus after Anti-biotic fermentation (CAB), the inventors have discovered that the FW are rich in saccharide and fiber which turns to be ideal substrate for bioconversion. Therefore, the combination of these bio-wastes can be used in bio-butanol production, and can replace the starch substrate and vitamin additives in fermentation culture medium.

As used herein, "DSG" means the Malt wine Distiller's spent grains As used herein, "POME" means the Palm oil mill effluent.

As used herein, "CAB" means the Cellulus after Anti-biotic fermentation. CAB is the centrifugation residue collected after antibiotic fermentation. This consists mainly of cell mass of mycelium, such as Cephalosporium (from cephalosprorin fermentation), Penicillium

(from penicillins fermentation), Saccharopolyspora (from Erythromycin fermentation) or Streptomyces (from Terramycin fermentation). This cell mass contains about 70% of protein

(dry weight) which makes it an ideal nitrogen source. However, it could not be used as animal feedstock because of residues of antibiotics, and they have to be treated and disposed of carefully as waste. The invention also provides the utilization of this nitrogen source as a substrate or an additive (co-substrate) in Clostridium fermentation to produce useful products such as solvents (acetone, butanol, and ethanol) as well as hydrogen gas.

As used herein, "FW" means the Food waste. Usually, FW is grinded to syrup for use. As used herein, "CSS" means the Corn stem stock. As used herein, "WGS" means the Wheat and Grain straw. As used herein, "BW" means Brewery waste.

The invention provides novel Bio-wastes which can be use as fermentation substrates in high efficiency, especially as substrates for Clostidium.

One ideal fermentation substrate is Residues after Anti-biotic fermentation (CAB). Another ideal fermentation substrate is Food waste (FW), which consists of raw vegetable and fruit wastes, including potatoes, cabbage, strawberry, orange peel, banana, carrot, apple, lettuce leaves, cauliflower leaves, etc.

The invention further provides a series of combination of different bio-wastes, which includes but is not limited to:

Malt wine Distiller's spent grains (DSG), solid, with 30-45% water Palm oil mill effluent (POME), liquid

Cellulus after Anti-biotic fermentation (CAB), cake, with 35-70% water Food waste (FW), consist of raw vegetable and fruit wastes, including potatoes, cabbage, strawberry, orange peel, banana, carrot, apple, lettuce leaves, cauliflower leaves, etc. Corn stem stock (CSS), solid, with 8-20% water Wheat and Grain straw (WGS), solid, with 8-20% water Brewery waste (BW), solid, with 30-45% water.

Different wastes are mixed to be substrate, the recipe could be but not limited to: DSG+CAB

POME+CAB WGS+BW FW+CSS CSS+BW POME+FW

CSS+DSG

The combination of the different wastes can be mixed by 2 to 4 different wastes at any ratio.

This invention also provides the production method using the above-mentioned bio-wastes as substrates, including the following reaction steps:

a) Pre-treatment of substrate

For example, CAB, dried at 80 centigrade, to control the water content less than 8%, grinded to powder at 100 mesh . b) chemical treatment of substrate

The CAB is usually prepared by the following steps: centrifuging the anti-biotic fermentation broth, thereby obtaining bacterial precipitate; treating said bacterial precipitate with 0.01-lN HNO₃, and adjusting pH to 4.0-8.0. For example, 100 grams of CAB (dry and grinded to powder), add 500 ml 0.16 M

HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to nuetralisation, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Add cellulose to hydrolyte the substrate, at 35-50 Centigrade, 150-250 rpm agitation, 8-18 hours. Then centrifuge to remove the insoluble residue. Collect the supernatant as saccharide culture medium. c) Fermentation

Inoculate 100 ml of the working culture anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically. Detect the production of Butanol, Ethanol, and Acetone in the cell culture by standard GC method.

Combination of different bio-wastes as substrate

The bio-wastes in this invention can be used alone or with other ones. Different wastes are mixed to be substrate, the recipe could be but not limited to:

DSG+CAB

POME+CAB

WGS+BW

FW+CSS CSS+BW

POME+FW

CSS+DSG

Advantages of this invention including:

(a) Do not use corps as substrate. Only use bio-waste as substrate, thus reduce the cost of raw materials dramatically

(b) Use bio-waste as substrate. It is also a new method to treat the environment contamination. For example, the CAB is very difficult to deal with after production of anti-biotic, because of its high protein contents (High BOD) and its residue of anti-biotic which limited its use as cattle feed.

(c) The fermentation time is shortened.

The invention is further illustrated by the following examples. It is appreciated that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples, they are performed under routine conditions, e.g., those described by Sambrook et al., in Molecule Clone: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturers, unless otherwise specified.

Example 1 choice of substrates

One of the following wastes is chosen to be the substrate: Malt wine Distiller's spent grains (DSG), solid, with 30-45% water Palm oil mill effluent (POME), liquid

Cellulus after Anti-biotic fermentation (CAB), cake, with 35-70% water

Food waste (FW), consist of raw vegetable and fruit wastes, including potatoes, cabbage, strawberry, orange peel, banana, carrot, apple, lettuce leaves, cauliflower leaves, etc.

Corn stem stock (CSS), solid, with 8-20% water

Wheat and Grain straw (WGS), solid, with 8-20% water

Brewery waste (BW), solid, with 30-45% water.

POME+CAB

WGS+BW

FW+CSS

CSS+BW POME+FW

CSS+DSG

Example 2 Pre-treatment of DSG

DSG, dried at 80 centigrade, to control the water content less than 10%, hammer-milled to powder.

POME, use directly.

Example 3 Pre-treatment of CAB

CAB, dried at 80 centigrade, to control the water content less than 8%, grinded to powder at 100 mesh.

The CAB in Example 3 is centrifugation residue collected after antibiotic fermentation, and includes Cephalosporium (from cephalosprorin fermentation), Penicillium (from penicillins fermentation), Saccharopolyspora (from Erythromycin fermentation) or Streptomyces (from Terramycin fermentation).

Example 4 Pre-treatment of FW

FW, grinded to syrup for use. Example 5 Pre-treatment of CSS

CSS, dried at ambient temperature, control the water content less than 10%, grinded to powder at 100 mesh .

Example 6 Pre-treatment of WGS

WGS, dried at ambient temperature, control the water content less than 10%, grinded to powder at 100 mesh .

Example 7 Pre-treatment of BW

BW, dried at 80 centigrade, to control the water content less than 8%, grinded to powder at 100 mesh .

Example 8 Chemical treatment of DSG 100 grams of DSG (after treatment in Example 2), add 500 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to neutralization, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Add cellulose to hydrolyte the substrate, at 35-50 Centigrade, 150-250 rpm agitation, 8-18 hours. Then centrifuge to remove the insoluble residue. Collect the supernatant as saccharide culture medium.

Example 9 Chemical treatment of CAB

100 grams of CAB (after treatment in Example 3), add 500 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to nuetralisation, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Example 10 Chemical treatment of FW

200 grams of FW (after treatment in Example 4), add 400 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to neutralization, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Example 11 Chemical treatment of CSS 100 grams of CSS (after treatment in Example 5), add 500 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to neutralization, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Example 12 Chemical treatment of WGS

100 grams of WGS (after treatment in Example 6), add 500 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to neutralization, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Example 13 Chemical treatment of BW

100 grams of BW (after treatment in Example 7), add 500 ml 0.16 M HNO₃, agitation at room temperature for 12 hours. Use 10 M NaOH to neutralization, adjust pH to 4.0-7.8; more preferable, the pH value between 5.0-6.8; and the best, the pH value between 5.5-6.8.

Example 14 Recovery of cell line

Clostridium acetobutylicum (ATCC 824) was maintained as a spore suspension at 4 centigrade. Spores are treated at 80 centigrade for 10 minutes for heat-shock, and then inoculated into 10 ml of reinforced clostridial medium, incubate at 37 centigrade for 14 hours in an anaerobic cabinet under N₂-H₂ (90-10) atmosphere.

Example 15 Preparation of working culture The clostridial basal medium (CBM) contains:

Carbon source 1Og

Casein hydrolysate 4g

KH₂PO₄ 0.5g

K₂HPO₄ 0.5g

MgSO₄*7H₂O 0.2g

MnSO₄*4H₂O lOmg

NaCl lOmg

FeSO₄*7H₂O lOmg p-aminobenzoic acid lmg thiamine HCl lmg d-biotin 2ug

Add double distilled water to make 1 liter, autoclaved at 121 centigrade for 20 minutes. Inoculate 5 ml the overnight culture (from Example 14) anaerobically to 100 ml clostridial basal medium + 6% glucose.

Incubate at 37 centigrade anaerobically for 20 hours

Example 16 Fermentation 1

Culture medium: CBM (prepared in Example 15) 3500 ml

DSG (prepared in Example 8) 1500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Detect the production of Butanol, Ethanol, and Acetone in the cell culture by standard GC method.

Example 17 Fermentation 2

Culture medium:

CBM (prepared in Example 15) 3500 ml CAB (prepared in Example 9) 1500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically. Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically. Detect the production of Butanol, Ethanol, and Acetone in the cell culture by standard GC method.

Example 18 Fermentation 3

Culture medium: CBM (prepared in Example 15) 500 ml

POME (prepared in Example 1) 4500 ml

Example 19 Fermentation 4

Culture medium:

CBM (prepared in Example 15) 3500 ml WGS (prepared in Example 12) 1500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 20 Fermentation 5

Culture medium:

CBM (prepared in Example 15) 3500 ml

FW (prepared in Example 10) 1500 ml Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 21 Fermentation 6

Culture medium:

CBM (prepared in Example 15) 3500 ml

CSS (prepared in Example 11) 1500 ml

Example 22 Fermentation 7

Culture medium:

CBM (prepared in Example 15) 3500 ml

BW (prepared in Example 13) 1500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically. Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 23 Fermentation 8 Culture medium:

CBM (prepared in Example 15) 3000 ml

CAB (prepared in Example 9) 500 ml

DSG (prepared in Example 8) 1500 ml

Example 24 Fermentation 9 Culture medium:

CBM (prepared in Example 15) 0 ml

CAB (prepared in Example 9) 500 ml

POME (prepared in Example 1) 4500 ml

Example 25 Fermentation 8 Culture medium:

CBM (prepared in Example 15) 3000 ml

CAB (prepared in Example 9) 500 ml

WGS (prepared in Example 12) 1500 ml Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Example 26 Fermentation 9

Culture medium:

CBM (prepared in Example 15) 3000 ml

CAB (prepared in Example 9) 500 ml

Example 27 Fermentation 10

Culture medium:

CBM (prepared in Example 15) 3000 ml

CAB (prepared in Example 9) 500 ml

CSS (prepared in Example 11) 1500 ml Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Example 28 Fermentation 11

Culture medium:

CBM (prepared in Example 15) 3000 ml

CAB (prepared in Example 9) 500 ml

BW (prepared in Example 13) 1500 ml Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 29 Fermentation 12

Culture medium:

CBM (prepared in Example 15) 3500 ml

FW (prepared in Example 10) 750 ml CSS (prepared in Example 11) 750 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 30 Fermentation 13

Culture medium:

CBM (prepared in Example 15) 3500 ml

CSS (prepared in Example 11) 1500 ml BW (prepared in Example 13) 500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Example 31 Fermentation 14

Culture medium:

CBM (prepared in Example 15) 2000 ml

POME (prepared in Example 1) 1500 ml FW (prepared in Example 10) 1500 ml

Inoculate 100 ml of the working culture (prepared in Example 15) anaerobically.

Incubate at pH 5.5-6.5, 37 centigrade for 60-110 hours anaerobically.

Detect the production of Butanol, Ethanol, and Acetone in the cell culture by standard GC method. Example 32 Fermentation 15

Culture medium: CBM (prepared in Example 15) 3000 ml CSS (prepared in Example 11) 1500 ml DSG (prepared in Example 8) 500 ml

Example 33 GC detection results of fermentation

The results of Examples 16-32 are shown in the following Table.

* A large amount of H₂ was produced. The results show that,

(1) For H2 production, CAB is an ideal substrate.

(2) For butanol production, CAB is not the ideal substrate if used separately and solely, but it does raise the growth rate of Clostridium, which obviously shorten the fermentation time of production.

(3) POME can be used solely as substrate, but contributes low organic solvent productivity; but when it used in combination with FW, its shows very high productivity and can shorten the fermentation time as well. (4) For butanol production, it is preferred to add C resource in addition to CAB.

It also should be understood that non-innovative modification or changes made by other technical staff in this field after reading this invention fall within claims of this invention as well.

Claims

1. A use of the residues from anti-biotic fermentation or CAB as the N resource in the Clostridium fermentation.

2. A culture medium useful for Clostridium fermentation wherein said culture medium contains the residues from anti-biotic fermentation or CAB as N resource.

3. The culture medium of Claim 1 wherein said culture medium further contains a C resource selected from the group consisting of Malt wine Distiller's spent grains, Palm oil mill effluent (POME), Food waste (FW), Corn stem stock (CSS), Wheat and Grain straw (WGS), Brewery waste (BW), sugar, starch, and the combinations thereof.

4. The culture medium of Claim 3 wherein the ratio of said CAB and said C resource by dry weight is from 100:50 to 100:500.

5. A method of Clostridium fermentation comprising the step of

6. The method of Claim 5 wherein the said fermentation condition further includes the presence of a C resource selected from the group consisting of Malt wine Distiller's spent grains, Palm oil mill effluent (POME), Food waste (FW), Corn stem stock (CSS), Wheat and Grain straw (WGS), Brewery waste (BW), sugar, starch, and the combinations thereof.

7. The method of Claim 6 wherein the ratio of said CAB and said C resource by dry weight is from 100:50 to 100:500.

8. The method of Claim 5 wherein the method further comprises the step of :

9. A method for producing bio-fuel by Clostridium fermentation comprising the following step(s):

10. A use of the residues from anti-biotic fermentation or CAB in the preparation of culture medium.