TWI604055B - Process for producing butanol - Google Patents

Description

Method for producing butanol

The present invention relates to a method for producing butanol, which comprises: immobilizing a strain of Clostridium spp. on an immobilization carrier; and fixing the strain The strain of the Clostridium species is subjected to continuous fermentation to cause butanol to be produced, wherein butanol is used during in-situ gas stripping by using a carrier gas during continuous fermentation. Be recycled.

Butanol has high energy density, low vapour pressure, low polarity, and miscibility with gasoline, and is therefore used as a fuel extender. And is considered to be one of the important energy sources in the future.

Butanol can be used by Clostridium spp. under an anaerobic condition [eg, Clostridium acetobutylicum , Clostridium beijerinckii , and Clostridium acetoacetate ) A strain of ( Clostridium saccharoperbutylacetonicum )] is produced by performing a fermentation reaction. However, butanol is cytotoxic, and when the butanol formed in the fermentation reaction is accumulated more, the growth of the strain is inhibited, which in turn leads to a decrease in the yield of butanol.

In order to reduce the adverse effects caused by the accumulation of butanol, it has been studied to continuously remove butanol by immobilized continuous fermentation or gas stripping. For example, in Qureshi N. et al . (2004), Appl. Biochem. Biotechnol ., 114:713-721, Qureshi N. et al. will use sterilized clay brick particles (clay) as an immobilization carrier. The brick particles were placed in a reactor, followed by inoculation of activated C. beijerinckii into the reactor, which was then filled with P2 medium. After 4 hours of culture, fresh P2 medium was continuously fed to the reactor, and the culture in the reactor was continuously discharged, at a dilution rate of 0.32 h ^-1 . Continuous fermentation is carried out. The experimental results show that the yield of butanol obtained by immobilized continuous fermentation is significantly better than that of batch fermentation.

In Ezeji TC et al . (2003), World J. Microbiol. Biotechnol ., 19:595-603, Ezeji TC et al . inoculated C. beijerinckii into a fermentation tank containing P2 medium for batch fermentation. At the 15th hour from the start of the batch fermentation, the CO ₂ and H ₂ generated during the fermentation were used as a carrier gas and the fermentation culture in the fermentation tank was carried out at a flow rate of 3 LPM. The in situ gas stripping is performed, and the extracted butanol is collected by a condenser. The experimental results show that stripping can improve the butanol yield and glucose utilization of batch fermentation.

In addition, in order to further improve the efficiency of stripping, it has been studied to remove the cells in the fermentation culture, and then to strip the obtained fluid-free portion. For example, CN 103555560 B discloses an apparatus for preparing butanol by acetone butanol fermentation coupling separation and purification, and a method for preparing butanol by using the apparatus, the method comprising: inoculating a butanol producing bacterium into a stirring bioreactor The fermentation is carried out, and the cells are separated from the fermentation culture by an immobilization device and a bacterial liquid separation device, and then the obtained fluid portion containing no cells is stripped. In the embodiment of this Continental patent, the stripping efficiency of the method proved to be significantly superior to the method without using the bacterial liquid separation device.

Master's thesis by Wang Yurong, Institute of Chemical Engineering, National Chung Hsing University {Name: "Continuously immobilized acetone butanol ethanol fermentation for in situ butanol removal directly from immobilized beds [Direct in situ butanol removal on In the packed bed during continuous and immobilized Acetone-Butanol-Ethanol (ABE) fermentation]"}, Wang Yurong uses fixed immobilized Clostridium acetobutylicum for continuous fermentation, and uses oleyl alcohol as an extraction solvent. The obtained fermentation culture was subjected to extraction to obtain an oleyl alcohol extract containing butanol, which was then stripped by nitrogen in an extraction tank.

However, these prior studies required additional separation steps and equipment to increase the cost required, without the practical application of the industry. Therefore, if we can develop a process with high butanol yield, simple operating procedures and low cost, it will be what we hope to achieve.

Summary of invention

Accordingly, the present invention provides a method for producing butanol, comprising: immobilizing a strain of a Clostridium species on an immobilized carrier; and subjecting the strain of the immobilized Clostridium species to continuous fermentation, Butanol is produced, wherein butanol is recovered during the continuous fermentation by in-situ stripping using a carrier gas.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to simplify the butanol fermentation process carried out by strains using Clostridium species and to improve the efficiency of the process, the Applicant has found through continuous research on the continuous fermentation with fixed strains and in situ stripping during continuous fermentation. Recovery of butanol can effectively improve glucose utilization and butanol yield.

Accordingly, the present invention provides a method for producing butanol, comprising: immobilizing a strain of a Clostridium species on an immobilized carrier; and subjecting the strain of the immobilized Clostridium species to continuous fermentation, Butanol is produced, wherein butanol is recovered during the continuous fermentation by in-situ stripping using a carrier gas.

As used herein, the term "strain of Clostridium spp." is intended to encompass all strains belonging to the Clostridium species capable of producing butanol, and strains suitable for use in the Clostridium species of the present invention include , but not limited to: Clostridium acetobutylicum , Clostridium saccharoperbutylacetonicum , Clostridium beijerinckii , Clostridium tyrobutyricum , Clostridium butyricum Clostridium butyricum ), and combinations of them. In a preferred embodiment of the invention, the strain of the Clostridium species is Clostridium acetobutylicum.

As used herein, the term "immobilizing" or "immobilization" means confining a living microorganism to a particular spatial region or carrier, thereby causing the fluid dynamics exhibited by the microorganism. The hydrodynamic characteristic is different from the surrounding environment and can maintain the activity of the microorganism so that it can be used repeatedly.

According to the present invention, immobilization of strains of Clostridium species can be carried out by techniques well known and customary to those skilled in the art. In this regard, reference may be made, for example, to Qureshi N. et al . (2004) (same as above) and to Wang Yurong of the National Institute of Chemical Engineering of National Chung Hsing University for master's thesis (same as above).

It can be understood that the operating conditions for immobilization are further changed depending on the strain of the Clostridium species used, the type of the immobilized carrier, the particle size, and the carrier filling rate, etc., in order to achieve the best. Immobilization effect. The choice of these operating conditions is routinely determined by those skilled in the art.

Preferably, the immobilization carrier is selected from the group consisting of: clay brick, zeolite, ceramic, bone char, resin, wood (wood) Chip), coke, rice husk, cotton, corn stalk, calcium alginate, silica gel, and combinations thereof. In a preferred embodiment of the invention, the immobilization carrier is a clay brick.

According to the invention, the particle size of the immobilized carrier falls within the range of 0.15 mm to 5 mm. In a preferred embodiment of the invention, the particle size of the immobilized support falls within the range of 0.84 mm to 2.38 mm.

As used herein, the term "carrier filling rate" means the ratio of the weight of the immobilized carrier to the volume of the medium in the fermentation tank. According to the present invention, the carrier filling ratio of the immobilized carrier falls within the range of 20% (w/v, g/L) to 40% (w/v, g/L). In a preferred embodiment of the invention, the carrier filling rate of the immobilized carrier is 20% (w/v, g/L).

According to the present invention, the immobilization of the strain of the Clostridium species is carried out under an anaerobic condition and a culture at a temperature ranging from 30 ° C to 40 ° C for 30 minutes to 120 minutes. In a preferred embodiment of the invention, the immobilization of the strain of the Clostridium species is carried out under an anaerobic condition and at 37 ° C for 30 minutes.

According to the present invention, the strain of the immobilized Clostridium species can be first subjected to batch fermentation before continuous fermentation to increase the number of strains of the immobilized Clostridium species.

In accordance with the present invention, the continuous fermentation can be carried out using techniques well known and customary to those skilled in the art. In this regard, reference may be made, for example, to Qureshi N. et al . (2004) (same as above) and to Wang Yurong of the National Institute of Chemical Engineering of National Chung Hsing University for master's thesis (same as above).

It can be appreciated that the operating conditions for continuous fermentation are further varied with factors such as the strain of Clostridium species used and the formulation of the medium to achieve optimal fermentation results. The choice of these operating conditions is routinely determined by those skilled in the art.

According to the present invention, the medium used for the continuous fermentation contains a carbon source, a nitrogen source, and an inorganic salt suitable for growth of a strain of Clostridium species. The selection of these carbon sources, nitrogen sources and inorganic salts falls within the professional literacy and routine technology of those skilled in the art.

According to the present invention, the continuous fermentation is carried out at a dilution rate ranging from 0.05 h ^-1 to 0.2 h ^-1 . In a preferred embodiment of the invention, the continuous fermentation is carried out at a dilution rate of 0.1 h ^-1 .

As used herein, the term "dilution rate" means the ratio of the feed rate to the volume of the fermentation culture in the fermentation tank.

According to the invention, the continuous fermentation is carried out under anaerobic conditions.

According to the present invention, the in situ gas stripping can directly remove the butanol produced by the strain of Clostridium species in the fermentation tank from the fermentation culture without first fermenting the culture Move to an extra unit. This in-situ stripping can be carried out using techniques well known and customary to those skilled in the art. In this regard, reference is made, for example, to Ezeji TC et al . (2003) (same as above).

It can be understood that the operating conditions for in-situ stripping are further varied with factors such as the type of carrier gas used and the butanol content to achieve the best extraction. The choice of these operating conditions is routinely determined by those skilled in the art.

Preferably, the carrier gas is selected from the group consisting of nitrogen, carbon dioxide, hydrogen, and combinations thereof. In a preferred embodiment of the invention, the carrier gas is nitrogen.

According to the invention, the in-situ stripping is carried out at a carrier gas flow rate ranging from 1 LPM to 4 LPM. In a preferred embodiment of the invention, the in-situ stripping is carried out at a carrier gas flow rate of 1 LPM. In a more preferred embodiment of the invention, the in-situ stripping is carried out at a carrier gas flow rate of 2 LPM.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting. EXAMPLES General Experimental Methods: 1. Gas chromatography (GC) analysis:

In the following examples, the concentration of butanol contained in each sample to be tested was measured using a gas chromatograph (GC) (Hewlett Packard HP 5890 Series II). The various operating conditions for the gas chromatograph are shown in Table 1 below. Table 1. Operating conditions of the gas chromatograph         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Operational Parameters</td><td> Conditions</td></tr><tr> <td> Separation column </td><td> DP-FFAP capillary column having a length of 30 m and an inside diameter of 0.32 mm, and using one with a 0.25 μm Film of thickness. </td></tr><tr><td> The temperature rise procedure of the column oven</td><td> is maintained at a starting temperature of 50 ° C for 4 minutes; then, the temperature is maintained It was raised to 100 ° C (20 ° C / min) and maintained at 100 ° C for 1 minute, then the temperature was raised to 170 ° C (30 ° C / min), and maintained at 170 ° C for 2.5 minutes. </td></tr><tr><td> carrier gas </td><td> nitrogen (N₂) </td></tr><tr> <td> Injector temperature </td><td> 225°C </td></tr><tr><td> Detector </td><td> Flame Discharge Detection Flame ionization detector (FID) </td></tr><tr><td> detector temperature </td><td> 225°C </td></tr></TBODY ></TABLE>

In addition, for comparison, different concentrations of butanol (purchased from Alfa Aesar) (2-30 g/L) were used as the control standard and the same analysis was performed. 2. Dinitrosalicylic acid (DNS) analysis:

First, 1 g of 3,5-dinitrosalicylic acid, 30 g of potassium sodium tartrate tetrahydrate, and 1.6 g of sodium hydroxide (sodium hydroxide) ) with 100 mL of deionized water to get a DNS reagent for use. Next, 1 mL of the sample to be tested was taken, and then 1 mL of the DNS reagent was added and mixed uniformly, and then the resulting mixture was placed in a water bath at 100 ° C for 10 minutes in the dark. Thereafter, the mixture was placed on ice for cooling, and then the absorbance (OD ₅₅₀ ) was measured with a spectrophotometer (SpectraMax M3, Molecular Devices) at a wavelength of 550 nm. The resulting OD ₅₅₀ absorbance value was converted to glucose content (g/L) based on a standard curve previously prepared with glucose having different known concentrations relative to their own OD ₅₅₀ absorbance values. Example 1. Continuous production of butanol with immobilized Clostridium acetobutylicum and in situ gas stripping : Experimental material : 1. Clostridium reinforced strain used in this example The reinforced clostridial medium (RCM) was purchased from Merck (Cat. No. 1.05411.0500). 2. The fermentation medium used in this example has a formulation as shown in Table 2 below. Table 2. Formulation of Fermentation Medium <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td>Ingredient</td><td> Concentration (g/L) </td></tr><tr><td> tryptone </td><td> 10 </td></tr><tr><td> NaCl </td><td> 10 </td></tr><tr><td> Yeast Extract</td><td> 5 </td></tr><tr><td>FeSO₄・7H₂O</td><td> 0.11 </td></tr><tr><td>MgSO₄・7H₂O</td><td> 0.6 </td></tr><tr><td>CaCl₂</td><td> 0.008 </td></tr><Tr><td>glucose</td><td> 60 </td></tr><tr><td> balance is deionized water</td></tr></TBODY></TABLE> 3. Preparation of inoculum of Clostridium acetobutylicum :

First, Clostridium acetobutylicum BCRC 10639, which is purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIDI) in Taiwan (corresponding to ATCC 824) was inoculated into RCM medium at a 5% (v/v) inoculum and subjected to a heat shock reaction for 2 minutes under an anaerobic condition and a water bath at 80 °C. Then, after cooling to room temperature, the culture was carried out under an anaerobic condition and in a constant temperature incubator (37 ° C, 150 rpm) for 24 hours to activate the strain, and the culture thus obtained was used as The inoculation source of Clostridium acetobutylicum in this example. 4. Prepare an immobilization carrier:

First, a hammer is used to break a clay brick purchased from a building material row, and then sequentially screened with a sieve having a diameter of 2.38 mm and 0.84 mm, thereby obtaining a range. Clay brick particles of particle size in the range of 0.84 mm to 2.38 mm. Thereafter, the clay brick granules were washed several times with deionized water, and then sterilized by a sterilizer (HVE-50 Autoclave, Hirayama) at 121 ° C and 1.2 atmospheres for 30 minutes. The sterilized clay brick particles thus obtained were used as the immobilization carrier in this example. 5. Continuous fermentation reactor:

The structure of the continuous fermentation reactor used in this example is shown in Fig. 1. The continuous fermentation reactor comprises a fermenter 1 (feeder 2), a feed port 2 connected to the fermenter 1, a discharge port 3, and a gas inlet 4 ( A gas inlet 4) and a gas outlet 5, and a condensing device 6 connected to the gas outlet 5. Fresh fermentation medium can be fed to the fermentation tank 1 via a feed port 2 for a butanol fermentation at a fixed flow rate, while the fermentation culture in the fermentation tank 1 will pass through the outlet at the same flow rate. The feed port 3 is discharged to the outside of the fermentation tank 1, so that the fermentation culture in the fermentation tank 1 is continuously replaced with fresh fermentation at a fixed rate [i.e., dilution rate]. The medium, and the contents in the fermentation tank 1, will be maintained at a fixed volume. A carrier gas for stripping can be introduced into the fermentation tank 1 via the gas inlet 4 at a fixed flow rate to extract butanol from the fermentation culture. The extracted butanol enters the condensing device 6 via the gas outlet 5 and is collected by the condensation of the condensing device 6. experimental method:

First, 420 g of sterilized clay brick particles obtained according to the fourth item "Preparation of immobilized carrier" of the above "Experimental Materials" were filled into the fermentation tank 1 of the continuous fermentation reactor. Next, 63 mL of the inoculation source of Clostridium acetobutylicum obtained according to the third item "Inoculation source of Clostridium acetobutylicum" in the above "Experimental Materials" was inoculated into the fermentation tank 1, and an anaerobic gas was used. The cells were cultured at 37 ° C for 30 minutes for cell immobilization. Thereafter, 2.1 L of the fermentation medium as shown in Table 2 above was added to the fermentation tank 1 so that the clay brick particles in the fermentation tank 1 had a filling of 20% (w/v, g/L). The filling rate was followed by batch fermentation of the fermentation tank 1 under an anaerobic condition, a temperature of 37 ° C, and a stirring rate of 50 rpm. The stirring rate was adjusted to 0 rpm from the 24th hour when the fermentation reaction was started. At the 48th hour from the start of the fermentation reaction, the fermentation culture was replaced with a fresh fermentation medium at a dilution rate of 0.1 h ^-1 , and the fermentation was continued until the fermentation reaction was started. The 288th hour is over.

During the continuous fermentation, the fermentation culture in the fermentation tank 1 is subjected to in-situ stripping according to the following conditions to continuously remove butanol from the fermentation culture: at the 48th to the 167th hour after the start of the fermentation reaction (under During the period of Phase 1), no stripping is performed; during the first 168 to 215 hours (hereinafter referred to as Stage 2) in which the fermentation reaction is started, stripping is performed using nitrogen gas having a flow rate of 1 LPM; During the first 216 to 288 hours (hereinafter referred to as stage 3) in which the fermentation reaction was started, stripping was carried out using nitrogen gas having a flow rate of 2 LPM.

During the continuous fermentation, sampling was carried out by collecting 10 mL of the fermentation culture from the discharge port 3, and the number of samplings during the stages 1, 2 and 3 was 16, 7 and 9 times, respectively. Sampling once every 7 to 8 hours. The fermentation culture collected by each sampling was centrifuged at 13,300 rpm for 5 minutes, and the obtained supernatant was subjected to the second item "Dinitrosalicylic acid analysis" according to the "general experimental method" above. The method described is used to measure the glucose content. The glucose utilization rate is calculated by substituting the glucose content contained in the measured fermentation culture into the following formula (1): Formula ( 1 ) : A = [( B - C) / B ] ×100 where: A = glucose utilization (%) B = glucose content of fermentation medium before fermentation (g / L) C = glucose content of fermentation culture (g / L) measured at each sampling time point

Next, the average glucose utilization rate (%) at each stage was further calculated. The experimental data obtained are expressed as mean ± standard deviation (Standard Deviation, S. D.).

Further, the supernatant obtained above was filtered with a needle filter having a pore size of 0.2 μm (purchased from Merck Millipore), and the obtained filtrate was subjected to the first item "gas phase according to the above "general experimental method". The method described in the chromatographic analysis was carried out to analyze the content of butanol. The butanol productivity is calculated by substituting the butanol content contained in the measured fermentation culture into the following formula (2): Formula ( 2 ) : D = E × F wherein: D = Butanol yield (g/Lh) E = butanol content of the fermentation culture (g/L) measured at each sampling time point F = dilution rate (h ^-1 )

Next, the average butanol yield (g/Lh) at each stage was further calculated. The experimental data obtained are expressed as mean ± standard deviation.

In addition, during the last 12 hours of Phase 2 (i.e., at the beginning of the 204th to 215th hours of the fermentation reaction) and during the last 12 hours of Phase 3 (i.e., during the 277th to 288th hours of the start of the fermentation reaction), Condensate 6 was used to collect the condensate, and 10 mL of condensate was collected after recording the volume of condensate collected for 12 hours. In addition, 10 mL of the fermentation culture was collected from the fermentation tank 1 at the end of Stage 2 and Stage 3, respectively. Next, the condensate and the fermentation culture were centrifuged at 13,300 rpm for 5 minutes, respectively, and the resulting supernatant was then filtered through a needle filter having a pore size of 0.2 μm. The filtrate obtained was analyzed for the content of butanol according to the method described in the first item "Gas Chromatography Analysis" of the "General Experimental Method" above. The butanol removal rate is calculated by substituting the butanol content contained in the measured condensate into the following formula (3): Formula ( 3 ) : G = (H × I) / J where: G = butanol removal rate (g / h) H = butanol content of condensate (g / L) I = volume of condensate (L) J = 12 (h)

The butanol selectivity is calculated by converting the measured condensate and the butanol content contained in the fermentation culture into a weight percentage and substituting the following formula (4): Formula ( 4 ) : K = [ L / ( 1- L )] / [ M / ( 1- M )] where: K = butanol selectivity L = weight percentage of butanol in the condensate (wt%) M = in the fermentation culture Butanol weight percentage (wt%) experimental results:

The results measured in this experiment are shown in Table 3 below. Table 3. Average Glucose Utilization, Average Butanol Yield, Butanol Removal Rate, and Butanol Selection Rate at Each Stage         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> stage</td><td> 1 </td><td> 2 </td> <td> 3 </td></tr><tr><td> Average glucose utilization (%) </td><td> 68.8±5.0 </td><td> 93.7±3.4 </td>< Td> 96.8±0.4 </td></tr><tr><td> average butanol yield (g/Lh) </td><td> 0.73±0.09 </td><td> 0.95±0.03 < /td><td> 0.96±0.06 </td></tr><tr><td> Butanol removal rate (g/h) </td><td> - </td><td> 0.525 < /td><td> 0.696 </td></tr><tr><td> Butanol selectivity </td><td> - </td><td> 21.8 </td><td> 24.1 < /td></tr></TBODY></TABLE>

As can be seen from Table 3, the average glucose utilization rates and average butanol yields in stages 2 and 3 were significantly higher than those in stage 1. The results of this experiment show that in the process of continuous fermentation, the use of nitrogen for in-situ stripping can significantly improve the utilization of glucose, thereby increasing the yield of butanol.

In addition, the butanol removal rate and the butanol selectivity of Stage 3 were significantly higher than those of Stage 2. The results of this experiment show that in-situ stripping with nitrogen at a flow rate of 2 LPM has a better butanol recovery utility.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Other features and advantages of the present invention will be apparent from the description of the drawings, wherein: Figure 1 is a schematic diagram of a continuous fermentation reactor.

1‧‧‧fermentation tank

2‧‧‧ Feed inlet

3‧‧‧Outlet

4‧‧‧ gas inlet

5‧‧‧ gas export

6‧‧‧Condensing device

Claims (8)

A method for producing butanol, comprising: immobilizing a strain of a Clostridium species on an immobilized carrier; and subjecting the strain of the immobilized Clostridium species to continuous fermentation to cause butanol to be produced Wherein butanol is recovered during the continuous fermentation by in-situ stripping using a carrier gas. The method of claim 1, wherein the immobilization carrier is selected from the group consisting of clay bricks, zeolites, ceramics, bone charcoal, resin, wood chips, coke, rice husks, cotton, corn stover, calcium alginate, Silicone, and combinations thereof. The method of claim 1, wherein the carrier filling rate of the immobilized carrier falls within a range of 20% (w/v, g/L) to 40% (w/v, g/L). The method of claim 1, wherein the carrier gas is selected from the group consisting of nitrogen, carbon dioxide, hydrogen, and combinations thereof. The method of claim 1, wherein the in-situ stripping is performed at a carrier gas flow rate ranging from 1 LPM to 4 LPM. The method of claim 1, wherein the strain of the Clostridium species is selected from the group consisting of Clostridium acetobutylicum , Clostridium saccharoperbutylacetonicum , and Bayesian Clostridium beijerinckii , Clostridium tyrobutyricum , Clostridium butyricum , and combinations thereof. The method of claim 1, wherein the continuous fermentation is carried out at a dilution rate ranging from 0.05 h ^-1 to 0.2 h ^-1 . The method of claim 1, wherein the continuous fermentation is carried out under an anaerobic condition.