CN114657215A - Method and device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria - Google Patents

Method and device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria Download PDF

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CN114657215A
CN114657215A CN202011527921.5A CN202011527921A CN114657215A CN 114657215 A CN114657215 A CN 114657215A CN 202011527921 A CN202011527921 A CN 202011527921A CN 114657215 A CN114657215 A CN 114657215A
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bacteria
butanol
lignin
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张全
吕明
高慧鹏
刘自勇
李澜鹏
范奕萱
乔凯
李福利
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China Petroleum and Chemical Corp
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Sinopec Dalian Research Institute of Petroleum and Petrochemicals
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China Petroleum and Chemical Corp
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Sinopec Dalian Research Institute of Petroleum and Petrochemicals
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Abstract

The invention provides a method and a device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria. The method comprises the following steps: (1) arranging sieve tray plates in the culture solution containing the enzymolysis corn straws or bagasse, and dividing the culture solution into an upper layer area and a lower layer area through the sieve tray plates; (2) inoculating activated aerobic lignin-degrading bacteria to the upper layer area in the step (1) for fermentation to obtain a fermentation liquor system; (3) inoculating the activated clostridium beijerinckii to the lower layer area in the step (1), and continuing to ferment and produce butanol. The invention carries out lignin degradation and butanol fermentation in the same system by a physical isolation mode, simplifies the process, simultaneously utilizes the lignin raw material, improves the yield of butanol and has important significance for the microbial industrial application of butanol.

Description

Method and device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a method and a device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria.
Background
Compared with fuel ethanol, the fuel butanol has the characteristics of high energy density and low volatility, and can improve the fuel efficiency and prolong the driving mileage; the engine does not generate SOx or NOx during combustion, has large mixing tolerance with gasoline, and does not need to modify the vehicle; less corrosive and suitable for use in existing fuel supply and distribution systems.
The process for producing butanol by industrial microorganisms is to take agricultural and sideline products such as starch, cassava and the like or sugar products such as molasses, sugarcane, beet and the like as raw materials, obtain sugar-containing water solution by pretreatment, inoculate butanol-producing strains (mostly anaerobic clostridium, such as clostridium beijerinckii and clostridium acetobutylicum) to perform Acetone Butanol (ABE) fermentation, and obtain n-butanol after rectification of fermentation liquor. The process equipment is similar to the production of fuel ethanol, the raw materials are cheap, the sources are wide, the investment is small, the fermentation condition is mild, and a precious metal catalyst is not needed.
The traditional biological butanol fermentation process is ubiquitous in that (1) the yield and the yield of butanol are low; (2) the final concentration of the solvent is low, and the energy consumption of the conventional rectification method is high; (3) the proportion of butanol in the total fermentation solvent is low, and the cost of butanol separation is high; (4) grain raw materials such as corn, molasses and the like are generally adopted for production, the cost of the raw materials is high, and the situation that grains compete with people exists.
Therefore, in order to reduce the raw material cost, the variety of fermentation raw materials is expanded, for example, agricultural wastes which are cheap and easy to obtain, such as corn stalks, bagasse, straws, defective fruits in orchards and the like, are utilized.
In the pretreatment method of lignocellulose such as corn straws, bagasse and the like, diluted acid, diluted alkali, ammonia explosion, steam explosion and other pretreatment processes are usually used, the lignocellulose raw materials can generate soluble lignin with a certain concentration, and the soluble lignin has toxic action on subsequent microbial fermentation, so that chemical and biological methods are developed to detoxify hydrolysate, the detoxification mechanism is researched, and the method has important significance for accelerating the fermentation efficiency and reducing the process cost.
Many studies of biomass degradation have demonstrated that: the first step in the lignin polymer degradation pathway is depolymerization to monolignols. There are three main lignin monomers, which are vanillin, p-coumaric acid and syringic acid. Wherein the intermediate metabolites of vanillin and syringic acid are guaiacol and catechol-dihydroxybenzene; the intermediate metabolite of p-coumaric acid and syringic acid is p-hydroxybenzoic acid, both chemical substances with benzene rings. Certain microorganisms can further ring-opening cleave the above-mentioned intermediate metabolites to produce low molecular weight acids (butyric, valproic, oxalic and propionic acids) and alcohols (ethanol and ethylene glycol).
CN 107760753A discloses a method for producing butanol by co-culture fermentation of high-temperature anaerobe for pyrolysis of sugar and clostridium acetobutylicum. Inoculating activated high-temperature anaerobic bacteria of the pyrolyzed sugar into a fermentation medium containing xylan for fermentation to obtain a fermentation liquid, and inoculating activated clostridium acetobutylicum into the fermentation liquid for fermentation to produce butanol. However, the invention does not relate to the degradation and conversion of lignin in complex raw materials (such as corn stalks).
In the existing technical approach for preparing butanol by industrial microbial fermentation, clostridium acetobutylicum and clostridium beijerinckii are mainly adopted, in order to reduce the cost of raw materials, lignocellulose raw materials such as corn straws, bagasse and the like are widely adopted, but soluble lignin is easily generated in the pretreatment process, and the lignin has toxic action on strains subjected to subsequent fermentation.
Disclosure of Invention
The invention aims to provide a method and a device for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria. Aiming at the agriculture and forestry wastes such as corn straws and corn residues and the like in the industrial microbial fermentation process of the biological butanol, the toxic action of the generated soluble lignin on the subsequent microbial fermentation in the pretreatment methods such as dilute acid, dilute alkali, ammonia explosion, steam explosion and the like is overcome.
The invention adds one or more aerobic lignin-degrading strains in the upper layer of a fermentation reaction system in enzymolysis saccharification hydrolysate of chemically (and/or physically) pretreated straws or bagasse. After a period of fermentation, anaerobic clostridium butyricum is added in the lower zone. The layered co-culture fermentation mode can reduce the lignin content in situ, and simultaneously the low molecular weight organic acid generated by metabolic conversion can further generate organic solvents such as acetone or butanol through an acid-alcohol conversion mechanism of clostridium, thereby reducing the consumption and cost of raw materials, simplifying the process, and improving the yield and the output of the organic solvents to a certain extent.
The invention provides a method for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria, which comprises the following steps:
(1) arranging sieve tray plates in the culture solution containing the enzymolysis corn straws or bagasse, and dividing the culture solution into an upper layer area and a lower layer area through the sieve tray plates;
(2) inoculating activated aerobic lignin-degrading bacteria to the upper layer area in the step (1) for fermentation to obtain a fermentation liquor system;
(3) inoculating the activated clostridium beijerinckii to the lower layer area in the step (1), and continuing to ferment and produce butanol.
The diameter of the holes of the sieve tray in the step (1) is between 300 and 1000 millimeters, and the number of the holes is between 40 and 100 per square meter; the sieve trays are positioned in the fermentation broth at a position 1/3-2/3 of their vertical height.
And (2) a stirring paddle is arranged in the lower layer area in the step (1). The upper layer area and the lower layer area in the step (1) are respectively as follows: in the whole fermentation liquid system, sieve trays are used as boundary lines, the region above the trays is the upper region, and the region below the trays is the lower region. Liquid between the upper layer area and the lower layer area can penetrate through the sieve holes to pass up and down, so that the fermentation system is uniform, but the rotating force of the lower layer stirring paddle can be offset through the physical blocking of the tower plates, and the repeated mixing of aerobic bacteria on the upper layer and anaerobic bacteria on the lower layer of the fermentation system is avoided.
The aerobic lignin degrading bacteria in the step (2) are one or more of the following bacteria: pseudomonas bacteria (Pseudomonas putida mt-2) with the accession number: DSMZ, DSM-6125, deposit date: in 1990, 7/month, disclosed in Journal of Bacteriology 115 (1) 262-267; streptomyces (I), (II)Streptomyces viridosporusT7A) having the accession number: ATCC 39115, date of deposit: 5.1981, published in Appl. environ. Microbiol. 42: 378-380; microbacterium (A), (B), (C)Microbacteriumsp) under the deposit accession number: 1.15785, preservation date: 9.2001, published in Biochemical Engineering journal 121(15), 2017, 131-; rhodococcus (A)Rhodococcus jostii RHA1) having the deposit accession number: DSM 44719, date of deposit: 2003, published in int.j.syst.evol.microbiol.52: 409-; burkholderia (B) ((B))Burkholderia cepacia) The preservation registration number is: DSM 50180, date of deposit: 1990, published in Enzyme and Microbial Technology 14 (11): 938-943; and Bacillus (A), (B)Alkalihalobacillus ligniniphilusL1, accession number: DSM 26145, deposit date: 2011, published in IntJ Syst Evol Microbiol 64 (Pt5): 1712-7. The clostridium beijerinckii in the step (3) is classified and named as clostridium beijerinckii (Clostridium beijerinckiiY-3) having the deposit accession number: CGMCC 5805, preservation date: published in CN102719371A in 2012.
Preferably, the fermentation conditions in step (2) are: the fermentation temperature is 20-40 ℃, the fermentation time is 12-50 h, the initial pH value of the fermentation is 5.0-7.5, and the rotating speed is 50-170 rpm.
Preferably, the concentration of the corn straws or the bagasse in the culture solution in the step (1) is 150-300 g/L. Preferably, the culture solution further comprises: 1-3 g/L sodium acetate, 0.5-3 g/L acetic acid, 1.0-5.0 g/L yeast powder, 0.2-1.0 g/L MgCl2·6H2O，0.1-0.6 g/L NH4Cl，0.01-0.05 g/L CaCl2·2H2O，0.5-2.0 g/L FeCl2·4H2O, 0.1-0.5 g/L KCl. Adjusting the pH value to 4.5-6.5.
The inoculation mode of the activated aerobic lignin-degrading bacteria in the step (2) is as follows: centrifuging the activated culture solution of the aerobic lignin-degrading bacteria, and inoculating the precipitate obtained by centrifugation into the upper layer area of the fermentation liquor system obtained in the step (1).
Preferably, the inoculation timing of clostridium beijerinckii in the step (3) is as follows: and (3) when the concentration of the soluble lignin in the fermentation liquor obtained in the step (2) is reduced to be less than 0.6 g/L, inoculating activated clostridium beijerinckii to the lower zone of the fermentation liquor obtained in the step (1), and fermenting to produce the butanol. When the concentration of the lignin is lower than 0.6 g/L, aerobic lignin degrading bacteria are enriched in the upper layer of the fermentation liquid, so that air isolation of the fermentation liquid can be realized, and fermentation of clostridium beijerinckii is carried out on the lower layer.
Preferably, the fermentation in step (1) is carried out in a fermentor. The adding proportion of the aerobic lignin degrading bacteria in the step (2) and the clostridium beijerinckii seeds in the step (3) is 1:1-1:3 in terms of volume ratio.
Preferably, the inoculation amount of the activated aerobic lignin-degrading bacteria in the step (1) is 1-20% of the total volume of the fermentation system.
Preferably, the activated anaerobic clostridium beijerinckii inoculation amount in the step (2) is 1-20% of the total volume of the fermentation system.
The fermentation conditions in the step (3) are as follows: the fermentation temperature is 35-40 ℃, the fermentation time is 60-168 h, the fermentation pH value is 4.5-7.0, and the rotation speed is 100-200 rpm.
In order to achieve the above objects, a second aspect of the present invention provides an apparatus for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria, comprising: the fermentation tank and the sieve tray arranged in the fermentation tank divide the fermentation tank into an upper layer area and a lower layer area through the sieve tray.
The diameter of the holes of the sieve tray is 300 mm-1000 mm, and the number of the holes is 40-100/square meter; the sieve trays are positioned in the fermentation broth at a position 1/3-2/3 of their vertical height.
And a stirring paddle is arranged in the lower layer area. The upper layer area and the lower layer area in the step (1) are respectively as follows: in the whole fermenter, the area above the tray is the upper zone and the area below the tray is the lower zone, with sieve trays as the dividing line. Liquid between the upper layer area and the lower layer area can penetrate through the sieve holes to pass through up and down, so that a fermentation system in the fermentation tank is uniform, but the rotating force of the lower layer stirring paddle can be offset through the physical blocking of the tower plates, and the repeated mixing of aerobic bacteria on the upper layer and anaerobic bacteria on the lower layer of the fermentation system is avoided.
Has the beneficial effects that: compared with the prior art, the invention has the following technical advantages:
(1) the invention carries out lignin degradation and butanol fermentation in the same system, simplifies the process, simultaneously utilizes the lignin raw material, improves the yield of butanol and has important significance for the microbial industrial application of butanol.
(2) According to the invention, aerobic lignin degrading bacteria capable of utilizing lignin are cultured firstly, soluble lignin in corn straw or bagasse enzymatic saccharification liquid is degraded and converted into low molecular weight acid (butyric acid, valproic acid, oxalic acid and propionic acid) and alcohol (ethanol and ethylene glycol) in the upper layer of a tower plate of a fermentation system, and then the soluble lignin is converted into organic solvents such as butanol and acetone through an acid-alcohol conversion mechanism in the fermentation process of clostridium beijerinckii at the lower layer of the fermentation system, so that the yield of the butanol is increased;
(3) when the aerobic lignin-degrading bacteria and the clostridium beijerinckii seeds are cultured according to the volume ratio of 1:2, the highest butanol yield reaches 12.92 g/L, and the solvent yield reaches 22.43 g/L, which is the highest butanol yield obtained by carrying out full utilization and co-culture on the components of the corn straws at present. When the aerobic lignin-degrading bacteria and the anaerobic microorganisms are co-cultured for 48 hours, the lignin concentration in the fermentation liquor is lower than 0.6 g/L, and 5 g/L of organic acid is obtained; then, in a co-culture system, the enriched aerobic lignin degrading bacteria can effectively block the oxygen content of the lower layer of the fermentation liquor, and the sieve pore tower plates can effectively isolate the growth areas of the aerobic bacteria and the anaerobic bacteria, so that the clostridium beijerinckii can continuously grow in the anaerobic environment;
(4) the method effectively reduces the cost of industrial production of butanol, solves the problem that the lignin in the straw or bagasse is difficult to utilize by dilute acid steam explosion, and has important application value.
Drawings
FIG. 1 is a schematic view of a co-culture fermentation system using aerobic bacteria and anaerobic bacteria according to the present invention;
wherein 1 is a fermentation liquor system, 2 is aerobic bacteria, and 3 is anaerobic bacteria;
FIG. 2 is the time-dependent concentration of the fermentation product and the substrate in example 1, in which the corn stalk enzymatic hydrolysate is used as the substrate;
FIG. 3 shows the butanol product and total solvent concentrations of fermentation broth in example 5 under different concentrations of pretreated corn stalk enzymatic hydrolysate;
FIG. 4 shows the concentration of butanol and total solvent produced by fermentation in the fermentation system of example 6 with different volume ratios of the upper and lower layers.
Detailed Description
FIG. 1 shows a schematic diagram of a fermentation system for co-culturing aerobic bacteria and anaerobic bacteria according to the present invention, wherein the fermentation system 1 is divided into an upper region (upper aerobic region) and a lower region (lower anaerobic region) by sieve trays (not shown). In the fermentation process, aerobic bacteria 2 are inoculated to the upper layer area, and anaerobic bacteria 3 are inoculated to the lower layer area. A stirring paddle is arranged in the fermentation system. The diameter of the holes of the sieve tray is 300 mm-1000 mm, and the number of the holes is 40-100/square meter; the sieve trays are positioned in the fermentation broth at a position 1/3-2/3 of their vertical height.
Example 1
Taking the corn straw enzymatic hydrolysate subjected to diluted acid steam explosion pretreatment as a carbon source, and utilizing Burkholderia (B) ((B))Burkholderia cepacia) And Clostridium beijerinckii: (C. beijerinckii: (C. beijerinckii))Clostridium beijerinckiiY-3) for co-cultivation fermentation to produce butanol (FIG. 1).
Selecting the strain Burkholderia (Bokholderia) from the plateBurkholderia cepacia) The single colony is inoculated into 20 mL of fermentation medium containing enzymolysis corn straw and cultured for 36h at 37 ℃ and 100 rpm. Then inoculating the strain to the upper layer area of the fermentation system by using the inoculum size of 10% v/v (taking the fermentation liquid system as the reference), stirring, culturing and fermenting at 37 ℃ and 100 rpm, wherein the initial fermentation pH value is 5.5, and obtaining the lignin removal and anaerobic detoxification fermentation liquid system in the lower layer area of the fermentation system.
Selecting the strain Clostridium beijerinckii from the plate (Clostridium beijerinckiiY-3) Single colonies were inoculated into 20 mL of fermentation medium and cultured at 37 ℃ for 36 hours at 100 rpm. When the concentration of soluble lignin in the fermentation liquor is reduced to<When the fermentation time is 36h, the clostridium beijerinckii is inoculated to the lower layer of the fermentation liquid by the inoculation amount of 10 percent v/v (based on the fermentation liquid system), the mixture is stirred and cultured for 72h at the temperature of 37 ℃ and the rpm of 100, and the fermentation pH is 4.5. Samples were taken every 24 hours from the reactor and the concentrations of the various products were determined by GC to give a final butanol yield of 12.92 g/L and an organic solvent yield of 22.43 g/L (FIG. 2).
The formula of the fermentation medium comprises 1.7 g/L of sodium acetate, 0.9 g/L of acetic acid, 4.5 g/L of yeast powder and 0.2 g/L of MgCl2·6H2O，0.15 g/L NH4Cl，0.02 g/L CaCl2·2H2O，0.5 g/L FeCl2·4H2O, 0.23 g/L KCl and 250g/L corn straw. Adjusting pH to 5.5, and sterilizing at 121 deg.C for 15 min.
Example 2
Taking corn straw enzymatic hydrolysate subjected to dilute acid steam explosion pretreatment as a carbon source, and utilizing pseudomonas (A), (B) and (C)Pseudomonas putidamt-2) and Clostridium beijerinckii: (Clostridium beijerinckiiY-3) fermenting to produce butanol.
Selecting the strain Pseudomonas from the plate (Pseudomonas putidamt-2) single colonies were inoculated into 10 mL of fermentation medium and cultured at 25 ℃ for 24h at 150 rpm. Then inoculating the strain to the upper layer area of a fermentation reaction system with the inoculation amount of 5% v/v, stirring, culturing and fermenting at 25 ℃, 150 rpm and the initial fermentation pH value of 6.5 to obtain the lignin removal and anaerobic detoxification fermentation liquid system of the lower layer area of the fermentation system.
Selecting the strain Clostridium beijerinckii from the plate (Clostridium beijerinckiiY-3) Single colonies were inoculated into 10 mL of fermentation medium and cultured at 35 ℃ for 24 hours at 150 rpm. When the concentration of soluble lignin in the fermentation liquor is reduced to<When the fermentation time is 48h, 0.6 g/L, the clostridium beijerinckii is inoculated to the lower layer of the fermentation reaction system with the inoculation amount of 10 percent v/v, the mixture is stirred and cultured for 150h at 35 ℃ and 150 rpm, and the fermentation pH is 5.0. Samples were taken from the reactor every 24 hours and the concentrations of the various products were determined by GC to give a final butanol yield of 10.97 g/L and an organic solvent yield of 20.68 g/L.
The formula of the fermentation medium comprises 1.5 g/L of sodium acetate, 0.8 g/L of acetic acid, 4.0 g/L of yeast powder and 0.30 g/L of MgCl2·6H2O，0.1 g/L NH4Cl，0.015 g/L CaCl2·2H2O，0.6 g/L FeCl2·4H2O, 0.18 g/L KCl and 250g/L corn straw. Adjusting pH to 6.5, and sterilizing at 121 deg.C for 15 min.
Example 3
Taking the corn straw enzymatic hydrolysate subjected to diluted acid steam explosion pretreatment as a carbon source, and utilizing bacillus (B), (B) and (C)Bacillus ligniniphilusL1 and Clostridium beijerinckii: (C. beijerinckii) ((C. beijerinckii))Clostridium beijerinckiiY-3) fermenting to produce butanol.
Selecting the strain Bacillus from the plate (Bacillus ligniniphilusL1) was inoculated into 20 mL of fermentation medium and cultured at 40 ℃ for 45 hours at 50 rpm. Then inoculating the strain to the upper layer of a fermentation reaction system by 5% v/v of the inoculation amount, stirring, culturing and fermenting at 40 ℃, 50 rpm and the initial fermentation pH value of 7.5 to obtain a lignin removal and anaerobic detoxification fermentation liquid system in the lower layer area of the fermentation system.
Selecting the strain Clostridium beijerinckii from the plate (Clostridium beijerinckiiY-3) single colonies were inoculated into 20 mL of fermentation medium and cultured at 40 ℃ for 36 hours at 100 rpm. When the concentration of soluble lignin in the fermentation liquor is reduced to<When the fermentation time is 0.6 g/L (25 h), the clostridium beijerinckii is inoculated to the lower layer of the fermentation reaction system in an inoculation amount of 10% v/v, the mixture is stirred and cultured for 100h at 40 ℃ and 100 rpm, and the fermentation pH is 6.5. Samples were taken from the reactor every 24 hours and the concentrations of the various products were determined by GC to give a final butanol yield of 11.76 g/L and an organic solvent yield of 21.32 g/L.
The formula of the fermentation medium comprises 1.7 g/L of sodium acetate, 0.9 g/L of acetic acid, 4.5 g/L of yeast powder and 0.2 g/L of MgCl2·6H2O，0.15 g/L NH4Cl，0.02 g/L CaCl2·2H2O，0.5 g/L FeCl2·4H2O, 0.23 g/L KCl and 200 g/L corn straw, adjusting the pH value to 7.5, and sterilizing at 121 ℃ for 15 min.
Example 4
Taking corn straw enzymatic hydrolysate subjected to dilute acid steam explosion pretreatment as a carbon source, and utilizing rhodococcus (R) ((R))Rhodococcus jostii RHA1 and Clostridium beijerinckii: (C.beijerinckii) ((C.beijerinckii))Clostridium beijerinckiiY-3) fermenting to produce butanol.
Selecting the strain Rhodococcus from the plate (Rhodococcus jostii RHA1) into 20 mL fermentation medium, culturing at 35 ℃ and 120rpm for 15h, then inoculating the single colony to the upper layer of the fermentation reaction system at an inoculation amount of 5% v/v, culturing and fermenting at 35 ℃ and 120rpm with stirring at an initial fermentation pH value of 6.0, and obtaining the lignin-removed and anaerobic detoxified fermentation liquid system in the lower layer region of the fermentation system.
Selecting the strain Clostridium beijerinckii from the plate (Clostridium beijerinckiiY-3) Single colonies were inoculated into 20 mL of fermentation medium and cultured at 37 ℃ for 36 hours at 100 rpm. When the concentration of soluble lignin in the fermentation liquor is reduced to<When the fermentation time is 36h, 0.6 g/L clostridium beijerinckii is inoculated to the lower layer of the fermentation reaction system with the inoculation amount of 10% v/v, the mixture is stirred and cultured for 72h at the temperature of 37 ℃ and the rpm of 200, and the fermentation pH is 5.5. Samples were taken every 24 hours from the reactor and the concentration of the various products was determined by GC, with a final butanol yield of 11.63 g/ml-L, the yield of the organic solvent is 20.97 g/L.
The formula of the fermentation medium comprises 1.7 g/L of sodium acetate, 0.9 g/L of acetic acid, 4.5 g/L of yeast powder and 0.2 g/L of MgCl2·6H2O，0.15 g/L NH4Cl，0.02 g/L CaCl2·2H2O，0.5 g/L FeCl2·4H2O, 0.23 g/L KCl and 275 g/L corn straw, adjusting the pH value to 6.0, and sterilizing at 121 ℃ for 15 min.
Example 5
Influence of different corn straw enzymatic hydrolysate concentrations on final butanol yield
(1) Aerobic lignin degrading microbe Buckholder: (Burkholderia cepacia) Inoculating the diluted acid steam explosion pretreated corn straw enzymatic hydrolysate system to the upper layer by an inoculation amount of 5% v/v, and performing activated culture at 37 ℃ and 120 rpm;
(2) activated Clostridium beijerinckii: (Clostridium beijerinckiiY-3) inoculating the corn straw enzymatic hydrolysate to the lower layer area of the corn straw enzymatic hydrolysate system with the inoculation amount of 5% v/v, fermenting at 37 ℃ and 120rpm for 96 hours to obtain fermentation liquor, and sampling and detecting every 24 hours;
the formula of the fermentation medium is as follows: 1.7 g/L sodium acetate, 0.9 g/L acetic acid, 4.5 g/L yeast powder, 0.2 g/L MgCl2·6H2O，0.15 g/L NH4Cl，0.02 g/L CaCl2·2H2O，0.5 g/L FeCl2·4H2O, 0.23 g/L KCl. Setting four groups of fermentation culture media, wherein the concentrations of the pretreated corn straws are respectively 150 g/L, 200 g/L, 250g/L and 300 g/L, adjusting the pH value to 5.5, and sterilizing at 121 ℃ for 15 min.
Sampling every 24h during fermentation to determine the butanol yield. When the concentration of the pretreated corn straws is 250g/L, the concentration of butanol obtained by co-culture is the highest, the yield of the butanol reaches 12.92 g/L, and the yield of the organic solvent reaches 22.43 g/L; when the concentration of the pretreated corn straws is 150 g/L, the concentration of butanol obtained by co-culture is only 8.53 g/L; when the concentration of the pretreated corn stalks is 300 g/L, the concentration of the butanol obtained by co-culture is 11.37g/L, which is slightly lower than that of the butanol obtained by co-culture when the concentration of the corn stalk enzymatic hydrolysate is 250g/L (figure 3).
Example 6
The effect of different ratios of aerobic to anaerobic microorganism seeds on butanol production.
The method is the same as example 1, except that: the proportion of the aerobic microorganism and the anaerobic microorganism (aerobic lignin-degrading bacteria, Burkholderia and anaerobic clostridium beijerinckii) seeds in the fermentation culture system is adjusted according to the volume ratio of (2: 1, 1:2 and 1: 3), and the concentration of the pretreated corn straw is 250 g/L.
Centrifuging the activated aerobic lignin degrading bacteria culture solution in the step (2) in a centrifuge at 4 ℃ at the rotating speed of 6000 rpm for 10 min, pouring out the supernatant, and adding bacteria mud into the upper layer of the fermentation liquor in the step (2), wherein the relation ratio of the volume of the activated aerobic lignin degrading bacteria culture solution to the volume of the fermentation liquor is 1:10, and the OD of the aerobic bacteria culture solution is600A value of 5 is reached; then continuing culturing at 37 ℃ and rotating at 100 rpm;
centrifuging the activated clostridium beijerinckii culture solution in the step (3) in a centrifuge at 4 ℃, rotating at 6000 rpm for 10 min, then pouring out the supernatant, adding bacterial sludge into the lower layer of the fermentation liquor in the step (3), wherein the ratio of the volume of the activated clostridium beijerinckii culture solution to the volume of the fermentation liquor is 2:10, and the OD of the anaerobic clostridium beijerinckii culture solution600A value of 5 is reached; then continuing culturing at 37 ℃, rotating at 100 rpm, controlling the pH value to be 5.5, and fermenting for 96 h;
sampling is carried out every 24h during the fermentation process of the butanol, and the butanol yield is determined. When the ratio of aerobic to anaerobic microorganism seed solutions was 1:2, the butanol yield was 9.84 g/L and the organic solvent yield was 19.68 g/L (FIG. 4).
Comparative example 1
Taking the corn straw enzymatic hydrolysate subjected to diluted acid steam explosion pretreatment as a carbon source, and utilizing Burkholderia (B) ((B))Burkholderia cepacia) And Clostridium beijerinckii: (Clostridium beijerinckiiY-3) co-culture fermentation to produce butanol.
Selecting the strain Burkholderia (Bokholderia) from the plateBurkholderia cepacia) Inoculating single colony into 20 mL fermentation culture medium containing enzymolysis corn stalk, culturing at 37 deg.C and 100 rpm for 36h, and inoculating 10% v/v (with fermentation liquid system)As reference) is inoculated into a fermentation system (no sieve tray is arranged in the fermentation system), and stirred and cultured at 37 ℃ and 100 rpm for fermentation to obtain a detoxified fermentation liquor system with lignin removed.
Selecting the strain Clostridium beijerinckii from the plate (Clostridium beijerinckiiY-3) single colony is inoculated into 20 mL fermentation medium, cultured at 37 ℃ and 100 rpm for 36h, then inoculated into the lower layer of fermentation liquor by an inoculum size of 10% v/v (based on the fermentation liquid system), stirred and cultured at 37 ℃ and 100 rpm for 72h, sampling is carried out in a reactor every 24 hours, the concentration of various products is measured by GC, and finally, the yield of butanol is not detected, and the yield of organic solvent is not detected because the clostridium beijerinckii cannot grow under the aerobic stirring environment.
The formula of the fermentation medium comprises 1.7 g/L of sodium acetate, 0.9 g/L of acetic acid, 4.5 g/L of yeast powder and 0.2 g/L of MgCl2·6H2O，0.15 g/L NH4Cl，0.02 g/L CaCl2·2H2O，0.5 g/L FeCl2·4H2O, 0.23 g/L KCl and 250g/L corn straw. Adjusting pH to 5.5, and sterilizing at 121 deg.C for 15 min.

Claims (15)

1. A method for producing butanol by co-culture fermentation of aerobic bacteria and anaerobic bacteria is characterized by comprising the following steps:
(1) arranging sieve tray plates in the culture solution containing the enzymolysis corn straws or bagasse, and dividing the culture solution into an upper layer area and a lower layer area through the sieve tray plates;
(2) inoculating activated aerobic lignin-degrading bacteria to the upper layer area in the step (1) for fermentation to obtain a fermentation liquor system;
(3) inoculating the activated clostridium beijerinckii to the lower layer area in the step (1), and continuing to ferment and produce butanol.
2. The process according to claim 1, wherein the sieve trays of step (1) have a diameter of between 300 mm and 1000 mm and a number of holes of between 40 and 100 holes per square meter; the sieve trays are positioned in the fermentation broth at a position 1/3-2/3 of their vertical height.
3. The method of claim 1, wherein a paddle is disposed in the lower zone in step (1).
4. The method of claim 1, wherein the aerobic lignin-degrading bacteria of step (2) are one or more of the following bacteria: pseudomonas bacteria (Pseudomonas putida mt-2), Streptomyces (Streptomyces: (Streptomyces)Streptomyces viridosporusT7A), Microbacterium (Microbacterium: (Microbacterium)Microbacteriumsp), Rhodococcus (Rhodococcus jostii RHA1), Burkholderia (B.))Burkholderia cepacia) And Bacillus (B), (B)Alkalihalobacillus ligniniphilusL1）。
5. The method according to claim 1, wherein the fermentation conditions of step (2) are: the fermentation temperature is 20-40 ℃, the fermentation time is 12-50 h, the initial pH value of the fermentation is 5.0-7.5, and the rotating speed is 50-170 rpm.
6. The method as claimed in claim 1, wherein the concentration of the corn stalks or the bagasse in the culture solution in step (1) is 150-300 g/L.
7. The method of claim 6, wherein the culture medium further comprises: 1-3 g/L sodium acetate, 0.5-3 g/L acetic acid, 1.0-5.0 g/L yeast powder, 0.2-1.0 g/L MgCl2·6H2O，0.1-0.6 g/L NH4Cl，0.01-0.05 g/L CaCl2·2H2O，0.5-2.0 g/L FeCl2·4H2O，0.1-0.5 g/L KCl。
8. The method of claim 1, wherein the activated aerobic lignin-degrading bacteria of step (2) are inoculated by the following method: centrifuging the activated culture solution of the aerobic lignin-degrading bacteria, and inoculating the precipitate obtained by centrifugation to the upper region of the fermentation liquid system obtained in the step (1).
9. The method according to claim 1, wherein the timing of the C.beijerinckii inoculation in step (3) is: and (3) when the concentration of the soluble lignin in the fermentation liquor obtained in the step (2) is reduced to be less than 0.6 g/L, inoculating activated clostridium beijerinckii to the lower zone of the fermentation liquor obtained in the step (1), and fermenting to produce the butanol.
10. The method according to claim 1, wherein the ratio of the aerobic lignin-degrading bacteria in step (2) to the clostridium beijerinckii seeds in step (3) is 1:1-1:3 by volume.
11. The method of claim 1, wherein the activated aerobic lignin-degrading bacteria of step (1) is inoculated in an amount of 1-20% of the total volume of the fermentation system.
12. The method of claim 1, wherein the activated anaerobic Clostridium beijerinckii of step (2) is inoculated in an amount of 1% to 20% of the total volume of the fermentation system.
13. The method according to claim 1, wherein the fermentation conditions of step (3) are: the fermentation temperature is 35-40 ℃, the fermentation time is 60-168 h, the fermentation pH value is 4.5-7.0, and the rotation speed is 100-200 rpm.
14. An apparatus for the fermentative production of butanol by co-cultivation of aerobic bacteria and anaerobic bacteria according to any of claims 1 to 13, comprising a fermenter and sieve trays arranged in the fermenter, wherein the fermenter is divided into an upper zone and a lower zone by the sieve trays.
15. The apparatus according to claim 14, wherein the sieve trays have a diameter of between 300 mm and 1000 mm and a number of holes of between 40 and 100 holes per square meter; the sieve trays are positioned in the fermentation broth at a position 1/3-2/3 of their vertical height.
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CN106479881A (en) * 2016-11-25 2017-03-08 清华大学 Prepare system and its application of butanol
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CN109423507A (en) * 2017-09-01 2019-03-05 中国科学院过程工程研究所 A kind of method of anaerobic-aerobic mixing fungin butanol solid state fermentation

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US5753474A (en) * 1995-12-26 1998-05-19 Environmental Energy, Inc. Continuous two stage, dual path anaerobic fermentation of butanol and other organic solvents using two different strains of bacteria
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