A method for producing medium-chain fatty acids from crop straw

Abstract

The present invention relates to a method for producing medium-chain fatty acids from crop straws, wherein the method first uses straws for simultaneous saccharification and co-fermentation to obtain electron donor ethanol; then uses Acetobacter pasteurianus to partially oxidize ethanol into electron acceptor acetic acid, and controls the fermentation time to adjust a suitable ethanol / acetic acid molar ratio; after solid-liquid separation, the liquid is used for carbon chain extension, the solid residue is pyrolyzed to prepare biochar, and the prepared biochar is used to strengthen the carbon chain extension process. Finally, pyrolysis carbon of straw fermentation residue is added to obtain a caproic acid concentration of 5.74 g / L, which is 2.3 times that of no addition. The method realizes the in-situ production of ethanol and acetic acid through two-step fermentation to provide electron donors and electron acceptors for carbon chain extension without exogenous addition; the preparation of biochar from straw fermentation solid residue effectively increases the caproic acid yield; the efficient and comprehensive utilization of all components of crop straw is achieved, and an economically feasible method is provided for the biorefining path of agricultural waste, which has industrial application value.

Description

Technical Field

[0001] The invention relates to the field of resource utilization of agricultural waste, and in particular to a method for producing medium-chain fatty acids from crop straws. Background Art

[0002] As a renewable resource, lignocellulosic organic waste has unique advantages in energy and chemical utilization. Bioconversion is the main way to utilize it, but traditional fermentation technologies represented by biogas and cellulosic ethanol always have common problems such as high cost and low product added value. Therefore, it is still necessary to explore the conversion path to produce high value-added products.

[0003] In recent years, with the rapid development of synthetic biology and biotechnology, a technology for synthesizing medium-chain fatty acids by carbon chain extension has become a research hotspot for the high-value utilization of wood fiber raw materials. Medium-chain fatty acids refer to straight-chain carboxylic acids with 6 to 12 carbon atoms, including hexanoic acid, heptanoic acid, decanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid, which are mainly used in organic synthesis, fragrances, medicines, lubricants, thickeners and biofuels. Generally, ethanol and acetic acid are commonly used electron donors and electron acceptors for carbon chain extension, and both ethanol and acetic acid can be obtained from wood fiber raw materials. The advantages of using wood fiber as a substrate source for medium-chain fatty acids are as follows: (1) Renewable biomass raw materials ensure the continuous supply of medium-chain fatty acid substrates for large-scale production; (2) Through multi-link design, electron donors and electron acceptors can be obtained from wood fiber raw materials at the same time, ensuring that the carbon chain extension process is carried out efficiently and stably. Compared with industrial acetic acid and ethanol, MCFAs (30,000 to 50,000 yuan / ton) have a higher market value. Therefore, converting cellulosic ethanol into medium-chain fatty acids with higher added value is of great significance for the large-scale resource utilization of wood fiber raw materials, especially straw.

[0004] CN 115976120 A discloses a method for producing medium-chain fatty acids by two-stage anaerobic fermentation of soybean dregs, CN112961884 A discloses a method for synthesizing medium-chain fatty acids from plantation waste, and CN 111394401 A discloses a method for preparing medium-chain fatty acids by anaerobic fermentation of wood fiber raw materials. In summary, it is found that most of the synthesis methods of medium-chain fatty acids require the additional addition of electron donor ethanol / lactic acid or electron acceptor acetic acid, while the use of commercial ethanol / acetic acid is not economical, and the process of adding substrates will also affect the stability of carbon chain extension. In addition, the solid residue after the fermentation of wood fiber raw materials is not effectively utilized.

[0005] The addition of exogenous media has a promoting effect on the carbon chain extension process, such as iron, phenol, charcoal, etc. CN113388648 A discloses a method for promoting anaerobic fermentation to produce medium-chain fatty acids and the efficiency of electron transfer between microorganisms by ferroferric oxide, and CN 111909970A discloses a method for enhancing the production of medium-chain fatty acids by anaerobic microbial fermentation by exogenous media. However, there is no report on the process of using the residual solids from straw fermentation to enhance the synthesis of medium-chain fatty acids. Summary of the invention

[0006] In view of the above, the present invention provides a method for producing medium-chain fatty acids from crop straw, which can overcome the defects of the prior art.

[0007] A method for producing medium-chain fatty acids from crop straws of the present invention comprises the following steps:

[0008] (1) Straw pretreatment: ultrafinely crushing the straw raw material to obtain pretreated raw material;

[0009] (2) Synchronous saccharification and co-fermentation: adding the pretreated straw material in step (1), cellulase, peptone, yeast extract, Saccharomyces cerevisiae cells and water into a reaction container for synchronous saccharification and co-fermentation, and controlling the solid content to be 20%;

[0010] (3) partial oxidation of ethanol: inoculating Acetobacter pasteurianus into the reaction vessel of step (2) for secondary fermentation;

[0011] (4) after step (3) is completed, solid-liquid separation is performed, the liquid is used for carbon chain extension, and the solid is dried for later use;

[0012] (5) Preparation of pyrolytic carbon from straw and fermentation residue: The solid dried in step (4) is pyrolyzed in a tubular furnace to prepare biochar;

[0013] (6) Carbon chain extension: The liquid portion obtained in step (4) is appropriately diluted and then nutrients (yeast extract, MgSO4·7H2O, K2HPO4 and (NH4)2SO4) are added. After adjusting the pH, Clostridium cohnii is inoculated and carbon materials are added to extend the carbon chain to obtain butyric acid and hexanoic acid.

[0014] In a specific case, the straw raw material in the above step (1) is at least one of corn straw, cotton straw, wheat straw or rice straw;

[0015] Preferably, in the above step (2), the amount of cellulase added is 15 FPU / g, peptone is 10 g / L, yeast extract is 5 g / L, the simultaneous saccharification and co-fermentation reaction temperature is 35° C., and the reaction time is 72 h.

[0016] Preferably, in the above step (3), the reaction temperature is 30° C., the reaction time is 0 to 48 h, and the preferred fermentation time is 24 h, i.e. the reaction is stopped when ethanol / acetic acid = 1:1 (molar ratio).

[0017] Preferably, the centrifugal parameters in the solid-liquid separation in the above step (4) are a rotation speed of 6500 rpm and a time of 10 min.

[0018] Preferably, the pyrolysis temperature in step (5) is 500-700° C., and the pyrolysis time is 60-120 min.

[0019] Preferably, in the above step (6), the liquid is diluted to a carbon molar concentration of 300 mM, the nutrients are added in the following amounts: 20 g / L yeast extract, 0.4 g / L MgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, the initial pH is adjusted to 6.5-7.0, and the carbon material used is one of granular activated carbon, powdered activated carbon, fermentation residue carbon and straw carbon.

[0020] In one embodiment, in step (6), the carbon chain extension process is enhanced by adding granular activated carbon, and the specific step is to add 15 g / L of granular activated carbon to the fermentation bottle to enhance the carbon chain extension process.

[0021] In another embodiment, in step (6), the carbon chain extension process is enhanced by adding powdered activated carbon, and the specific step is to add 15 g / L of powdered activated carbon to the fermentation bottle to enhance the carbon chain extension process.

[0022] In another embodiment, in step (6), the carbon chain extension process is enhanced by adding pyrolytic carbon from fermentation residues, and the specific step is to add 15 g / L pyrolytic carbon from fermentation residues into the fermentation bottle to enhance the carbon chain extension process.

[0023] In another embodiment, in step (6), the carbon chain extension process is enhanced by adding straw pyrolysis charcoal, and the specific step is to add 15 g / L of straw pyrolysis charcoal into the fermentation bottle to enhance the carbon chain extension process.

[0024] The advantages of the present invention are: (1) the electrons required for carbon chain extension are obtained by using crop straw through two-step fermentation in one pot, without the need for exogenous addition, thereby improving production efficiency while reducing costs, improving the feasibility of industrial production, and solving the problems of instability and economy of the fermentation system caused by the additional addition of ethanol / acetic acid in the existing process. (2) Biochar is prepared by using the solid residue left over from straw fermentation, which strengthens carbon chain extension, significantly increases the production of caproic acid, and solves the problem of insufficient utilization of the solid residue left over from fermentation; (3) A new model for comprehensive utilization of straw resources is established, giving full play to the advantages of utilizing all components of straw, with simple process operating conditions and low raw material costs, and promoting the added value of the agricultural industry through the high-value utilization of agricultural residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The features and advantages of the present invention will become more apparent with reference to the detailed description of the following drawings:

[0026] Figure 1 Shown is a process flow chart of the method of the present invention.

[0027] Figure 2 Shown is the curve of ethanol and acetic acid concentration changes during the fermentation process of Acetobacter pasteurianus.

[0028] Figure 3 The caproic acid concentration results of Examples 1-5 without adding carbon material and with adding different carbon materials. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments.

[0030] The straw raw material used in the embodiment of the present invention is corn straw, wherein the cellulose content is 32.6%, the hemicellulose content is 16.5%, and the lignin content is 16.6%, and it is crushed to a particle size of 1 mm by a crusher. The coarse straw is subjected to ultrafine powder processing by a nano impact mill, the volume ratio of the ball to the material is 2:1, and the ultrafine grinding time is 60 minutes.

[0031] Cellulase (Novozymes Cellic CTec3), wherein the enzyme activity of cellulase CTec3 was determined according to the determination method of NREL, and the enzyme activity was measured to be 240 FPU / mL.

[0032] The strains used were as follows:

[0033] (1) Saccharomyces cerevisiae WXY74, donated by the School of Life Sciences, Capital Normal University (C Zhang, et al., In-depth two-stage transcriptional reprogramming and evolutionary engineering of Saccharomyces cerevisiae for efficient bioethanol production from xylose with acetate. J. Agric. Food Chem. 2019, Vol. 67, 12002-12012). This strain was developed by introducing genes related to xylose metabolism in Pichia pastoris to enable Saccharomyces cerevisiae to utilize xylose. After four years of laboratory adaptive evolution, it can simultaneously utilize glucose and xylose to co-ferment ethanol. The seed culture medium was Yeast extract-Peptone-Dextrose medium (YPD liquid culture medium), including 20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract.

[0034] (2) Acetobacter pasteurianus BNCC 335801, purchased from Beijing Beina Chuanglian Biotechnology Research Institute. The culture medium consists of 10 g / L yeast extract, 10 g / L glucose and 0.5% anhydrous ethanol, and the anhydrous ethanol is added after the culture medium is sterilized and cooled to 50-60°C.

[0035] (3) Clostridium cohnii CICC 10388 was purchased from China Industrial Microbiological Culture Collection. The culture medium consisted of 10 g / L yeast extract, 5 g / L sodium acetate, 0.4 g / L K2HPO4, 0.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O and 2% anhydrous ethanol. The anhydrous ethanol was added after the culture medium was sterilized and cooled to 50-60°C.

[0036] Embodiment 1:

[0037] Simultaneous saccharification and co-fermentation: 4.5 g of ultrafinely crushed 60 min pretreated corn straw, 10 g / L peptone, 5 g / L yeast extract, 16 g of water, and 15 FPU / g of cellulase were added to a conical flask. Yeast cells (initial OD 600=2), set the reaction temperature to 35°C, the rotation speed to 200rpm, and the reaction time to 72h. Ethanol oxidation: inoculate 10% Acetobacter pasteurianus into the simultaneous saccharification and co-fermentation broth, set the reaction temperature to 30°C, the rotation speed to 200rpm, and the secondary fermentation time to 0-48h; solid-liquid separation: solid-liquid separation is performed after the secondary fermentation, the centrifugation parameters are the rotation speed of 6500rpm, the time is 10min, the solid is used for the preparation of biochar, and the liquid is used for carbon chain extension. The liquid obtained after solid-liquid separation was added to a serum bottle, and 20 g / L yeast extract, 0.4 g / LMgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4 were added at the same time. The initial pH was adjusted to 6.5-7.0, and the nitrogen was vented for 10 minutes. After sterilization, it was cooled to room temperature and inoculated with 10% Clostridium cohnii. The fermentation was carried out for 30 days. After gas chromatography, the butyric acid concentration was 1.80 g / L and the caproic acid concentration was 2.47 g / L.

[0038] Embodiment 2:

[0039] Simultaneous saccharification and co-fermentation: 4.5 g of ultrafinely crushed 60 min pretreated corn straw, 10 g / L peptone, 5 g / L yeast extract, 16 g of water, and 15 FPU / g of cellulase were added to a conical flask. Yeast cells (initial OD 600 =2), set the reaction temperature to 35°C, the rotation speed to 200rpm, and the reaction time to 72h. Ethanol oxidation: inoculate 10% Acetobacter pasteurianus into the simultaneous saccharification and co-fermentation broth, set the reaction temperature to 30°C, the rotation speed to 200rpm, and the secondary fermentation time to 0-48h; solid-liquid separation: solid-liquid separation is performed after the secondary fermentation, the centrifugation parameters are the rotation speed of 6500rpm, the time is 10min, the solid is used for the preparation of biochar, and the liquid is used for carbon chain extension. The liquid obtained after solid-liquid separation was added to a serum bottle, and 20 g / L yeast extract, 0.4 g / LMgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, and 15 g / L granular activated carbon (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added at the same time. The initial pH was adjusted to 6.5-7.0, and the nitrogen was vented for 10 minutes. After sterilization, it was cooled to room temperature and inoculated with 10% Clostridium coli. Fermentation was carried out for 30 days. After gas chromatography, the butyric acid concentration was 1.88 g / L and the caproic acid concentration was 3.85 g / L.

[0040] Embodiment 3:

[0041] Simultaneous saccharification and co-fermentation: 4.5 g of ultrafinely crushed 60 min pretreated corn straw, 10 g / L peptone, 5 g / L yeast extract, 16 g of water, and 15 FPU / g of cellulase were added to a conical flask. Yeast cells (initial OD 600=2), set the reaction temperature to 35°C, the rotation speed to 200rpm, and the reaction time to 72h. Ethanol oxidation: inoculate 10% Acetobacter pasteurianus into the simultaneous saccharification and co-fermentation broth, set the reaction temperature to 30°C, the rotation speed to 200rpm, and the secondary fermentation time to 0-48h; solid-liquid separation: solid-liquid separation is performed after the secondary fermentation, the centrifugation parameters are the rotation speed of 6500rpm, the time is 10min, the solid is used for the preparation of biochar, and the liquid is used for carbon chain extension. The liquid obtained after solid-liquid separation was added to a serum bottle, and 20 g / L yeast extract, 0.4 g / LMgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, and 15 g / L powdered activated carbon (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added at the same time. The initial pH was adjusted to 6.5-7.0, and the nitrogen was vented for 10 minutes. After sterilization, it was cooled to room temperature and inoculated with 10% Clostridium cohnii. Fermentation was carried out for 30 days. After gas chromatography, the butyric acid concentration was 1.97 g / L and the caproic acid concentration was 5.26 g / L.

[0042] Embodiment 4:

[0043] Simultaneous saccharification and co-fermentation: 4.5 g of ultrafinely crushed 60 min pretreated corn straw, 10 g / L peptone, 5 g / L yeast extract, 16 g of water, and 15 FPU / g of cellulase were added to a conical flask. Yeast cells (initial OD 600 =2), set the reaction temperature to 35°C, the rotation speed to 200rpm, and the reaction time to 72h. Ethanol oxidation: inoculate 10% Acetobacter pasteurianus into the simultaneous saccharification and co-fermentation broth, set the reaction temperature to 30°C, the rotation speed to 200rpm, and the secondary fermentation time to 0-48h; solid-liquid separation: solid-liquid separation is performed after the secondary fermentation, the centrifugation parameters are the rotation speed of 6500rpm, the time is 10min, the solid is used for the preparation of biochar, and the liquid is used for carbon chain extension. The liquid obtained after solid-liquid separation was added into a serum bottle, and 20 g / L yeast extract, 0.4 g / LMgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, and 15 g / L pyrolysis charcoal of straw fermentation residue were added at the same time. The initial pH was adjusted to 6.5-7.0, and the nitrogen was vented for 10 minutes. After sterilization, it was cooled to room temperature and inoculated with 10% Clostridium cohnii. Fermentation was carried out for 30 days. After gas chromatography, the butyric acid concentration was 1.98 g / L and the caproic acid concentration was 5.74 g / L.

[0044] The preparation method of straw fermentation residue pyrolysis carbon is as follows: the dried solid residue is placed in a tube furnace, the pyrolysis temperature is set to 500-700°C, the pyrolysis time is 60-120 minutes, and the residue is taken out after cooling to room temperature for use.

[0045] Embodiment 5:

[0046] Simultaneous saccharification and co-fermentation: 4.5 g of ultrafinely crushed 60 min pretreated corn straw, 10 g / L peptone, 5 g / L yeast extract, 16 g of water, and 15 FPU / g of cellulase were added to a conical flask. Yeast cells (initial OD 600 =2), set the reaction temperature to 35°C, the rotation speed to 200rpm, and the reaction time to 72h. Ethanol oxidation: inoculate 10% Acetobacter pasteurianus into the simultaneous saccharification and co-fermentation broth, set the reaction temperature to 30°C, the rotation speed to 200rpm, and the secondary fermentation time to 0-48h; solid-liquid separation: solid-liquid separation is performed after the secondary fermentation, the centrifugation parameters are the rotation speed of 6500rpm, the time is 10min, the solid is used for the preparation of biochar, and the liquid is used for carbon chain extension. The liquid obtained after solid-liquid separation was added into a serum bottle, and 20 g / L yeast extract, 0.4 g / LMgSO4·7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, and 15 g / L straw pyrolysis charcoal were added at the same time. The initial pH was adjusted to 6.5-7.0, and the nitrogen was vented for 10 minutes. After sterilization, it was cooled to room temperature and inoculated with 10% Clostridium cohnii. The fermentation lasted for 30 days. After gas chromatography, the butyric acid concentration was 1.86 g / L and the caproic acid concentration was 5.88 g / L.

[0047] The preparation method of straw pyrolysis carbon is as follows: the dried ball-milled straw is placed in a tube furnace, the pyrolysis temperature is set to 500-700°C, the pyrolysis time is 60-120 minutes, and the straw is taken out after cooling to room temperature for use.

[0048] Figure 2 The figure shows the concentration change curve of ethanol and acetic acid during the fermentation process of Acetobacter pasteurianus. Figure 2 It can be seen that as the fermentation time increases, ethanol is gradually oxidized to acetic acid. The ethanol / acetic acid (molar ratio) can be regulated by controlling the fermentation time. Calculation shows that when the fermentation time is 24h, ethanol / acetic acid (molar ratio) = 1:1.

[0049] Figure 3 The results of caproic acid concentration in Examples 1-5 without adding carbon materials and with adding different carbon materials. The concentrations of fatty acids (butyric acid and caproic acid) were determined by gas chromatography, and the analysis conditions were: nitrogen as the carrier gas, 30m×0.25mm×0.25mm DB-WAX as the detection column, split ratio 35:1; the detector was a hydrogen flame ionization detector, the vaporization chamber temperature was 280°C, and the FID temperature was 280°C; program heating was adopted: the column box started at 60°C and maintained for 2min; then, at 20°C / min, it was increased to 140°C, and then, it was increased to 150°C at 8°C / min and maintained for 1.5min, and then it was increased to 200°C at 120°C / min and maintained for 1.25min, and finally, it was increased to 240°C at 120°C / min and operated for 3min. The injection volume was 1.0μL each time. By Figure 3 It can be seen that compared with the control group (no carbon material added), the addition of different carbon materials can increase the caproic acid concentration. Due to the porosity and surface functional groups of the carbon materials themselves, a part of the caproic acid can be adsorbed, thereby reducing the effect of product inhibition on microbial growth. Among them, the caproic acid concentration of the straw fermentation residue pyrolysis carbon group and the straw pyrolysis carbon group was the highest, 5.74-5.88 g / L, significantly higher than the control group and the granular activated carbon group. In addition, the strengthening mechanism of carbon materials for carbon chain extension can be divided into the following aspects: (1) Carbon materials can colonize carbon chain extension microorganisms and play a certain protective role for microorganisms during the fermentation process; (2) Carbon materials adsorb caproic acid to reduce product inhibition; (3) The redox functional groups on the surface of carbon materials and the internal π electrons play an electron transfer role, promoting microbial growth metabolism and carbon chain extension process.

Claims

1. A method for producing medium-chain fatty acids from crop straw, characterized in that: The following steps are involved: (1) Straw pretreatment: Ultrafinely crush the straw raw materials to obtain pretreated raw materials; (2) Synchronous saccharification and co-fermentation: adding the pretreated straw raw material in step (1), cellulase, peptone, yeast extract, Saccharomyces cerevisiae WXY74 cells and water into a reaction container for synchronous saccharification and co-fermentation; (3) Partial oxidation of ethanol: inoculating Acetobacter pasteurianus BNCC 335801 into the reaction vessel of step (2) for secondary fermentation; (4) After step (3), solid-liquid separation is performed, the liquid is used for carbon chain extension, and the solid is dried for later use; (5) Preparation of pyrolysis carbon from straw and fermentation residue: The solid dried in step (4) is pyrolyzed in a tubular furnace to prepare biochar; wherein the pyrolysis temperature is 500-700 °C and the pyrolysis time is 60-120 min; (6) Carbon chain extension: diluting the liquid obtained in step (4), adding nutrients including yeast extract, MgSO4•7H2O, K2HPO4 and (NH4)2SO4, adjusting the pH, inoculating Clostridium cohnii CICC 10388 and adding the biochar material in step (5) to extend the carbon chain; the biochar material is 15 g / L straw pyrolysis charcoal or straw fermentation residue pyrolysis charcoal; In step (2), the solid content was controlled to be 20%, the amount of cellulase added was 15 FPU / g, the peptone was 10 g / L, the yeast extract was 5 g / L, the simultaneous saccharification and co-fermentation reaction temperature was 35 °C, and the reaction time was 72 h; In step (3), the reaction temperature is 30 °C, the reaction time is 0-48 h, and the reaction is stopped when the molar ratio of ethanol to acetic acid is 1:1; In step (6), the liquid is diluted to a carbon molar concentration of 250-350 mM, and the nutrients are added in the following amounts: 18-22 g / L yeast extract, 0.2-0.6 g / L MgSO4•7H2O, 0.6-1.0 g / L K2HPO4 and 0.8-1.2 g / L (NH4)2SO4, and the initial pH is adjusted to 6.0-7.5; Step (6) carbon chain extension to obtain butyric acid and / or hexanoic acid.

2. The method according to claim 1, characterized in that: In step (1), the straw raw material is at least one of corn straw, cotton straw, wheat straw or rice straw.

3. The method according to claim 1, characterized in that In step (4), the centrifugal parameters for solid-liquid separation are a rotation speed of 6500 rpm and a time of 10 min.

4. The method according to claim 1, characterized in that In step (6), the liquid was diluted to a carbon molar concentration of 300 mM, and the nutrients were added as follows: 20 g / L yeast extract, 0.4 g / L MgSO4•7H2O, 0.8 g / L K2HPO4 and 1.0 g / L (NH4)2SO4, and the initial pH was adjusted to 6.5~7.0.

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