Method for modifying and enhancing efficiency of microorganisms for producing caproic acid by using rice straws based on semiconductor material

Abstract

The invention discloses a method for modifying and enhancing the hexanoic acid production efficiency of microorganisms by using rice straws based on a semiconductor material, and relates to the technical field of preparation of hexanoic acid by microorganisms. Comprising the following steps: 1, pretreating rice straws; 2, constructing a hybrid system of the biological enhancement strain C.kluyveri-CdS, and carrying out biological enhancement on the strain C.kluyveri-CdS; 3, preparing an acetic acid-butyric acid mixed flora; 4, culturing the C.kluyveri-CdS and the mixed flora by taking the rice straw as a raw material; and 5, carrying out caproic acid fermentation. According to the method, the pretreated rice straw is used as a raw material, a C.kluyveri-CdS hybrid system is used as a biological enhancement strain, and photoelectrons generated by photoelectric properties of a semiconductor material of the C.kluyveri-CdS hybrid system are used as electron donors, so that the use of ethanol is avoided, and acetic acid and butyric acid are produced by anaerobic fermentation flora taking the pretreated rice straw as the raw material; the utilization rate of a substrate and the yield of hexanoic acid can be effectively improved, and the yield of hexanoic acid can reach 14.6 g / L. The method achieves the purpose of reducing extra addition of ethanol, acetic acid and butyric acid so as to reduce the production cost.

Description

Technical Field

[0001] The invention relates to a method for preparing caproic acid by microorganisms. Background Art

[0002] The current world's industrial chemicals are still mainly derived from fossil fuels, which leads to excessive carbon emissions and climate change, and raises sustainable development issues. It is clear that chemicals need to be manufactured in an environmentally sustainable and affordable way. Therefore, environmentally friendly bio-based products have received widespread attention. Hexanoic acid has the advantages of being green, low energy consumption, high density, and high added value, and can be used in industrial production such as food additives, medicines, and spices. The preparation methods of hexanoic acid include chemical synthesis and extraction. Industrial hexanoic acid production is mainly synthesized by chemical synthesis, that is, hexanoic acid is obtained by oxidizing 2-octanol with concentrated nitric acid, but this method has high energy consumption and heavy pollution, which is not conducive to environmental and economic sustainable development. Although hexanoic acid can be obtained from crops such as palm and coconut by extraction, the hexanoic acid concentration is low and the cost is high, so it is not widely used. In recent years, research on the preparation of bio-caproic acid by microbial fermentation has attracted much attention at home and abroad. This technology is based on the metabolism of microorganisms to synthesize bio-caproic acid from short-chain fatty acids through a carbon chain extension process. It has the advantages of being green and environmentally friendly, and is considered to be a promising method for preparing hexanoic acid. At present, the main limiting factor of producing caproic acid by microbial fermentation is that ethanol must be added as an electron donor and acetic acid, butyric acid and other substrates must be added as electron acceptors, which greatly increases the production cost. Using cheap lignocellulosic biomass as raw material is one of the effective ways to reduce production costs.

[0003] In the existing research on the production of energy materials by fermentation of lignocellulose, cellulase is generally used to hydrolyze lignocellulose into monosaccharides such as glucose and xylose, and then microbial fermentation is used to produce short-chain fatty acids such as acetic acid and butyric acid. In addition, the biohybrid system that combines the efficient light-collecting ability of inorganic semiconductors with the biosynthetic ability of microorganisms has received widespread attention as a sustainable and efficient chemical synthesis platform. The metal sulfide semiconductor material CdS is a visible light semiconductor photocatalytic material. It has a good photoelectric effect on visible light because of its narrow band gap and high electron-hole pair generation rate and mobility. It is widely recognized as a very promising photocatalytic material. The photoelectrons generated by the internal electron transition of CdS under visible light can be used as an electron donor to replace ethanol, thereby reducing production costs. Summary of the invention

[0004] The purpose of the present invention is to solve the problems of high cost of raw materials for caproic acid fermentation and the need to add ethanol as an electron donor at this stage, and to provide a method for enhancing the efficiency of microorganisms in producing caproic acid using rice straw based on semiconductor material modification.

[0005] The present invention discloses a method for enhancing the efficiency of microorganisms in producing caproic acid by utilizing rice straw based on semiconductor material modification, which is carried out according to the following steps: first, pretreating rice straw with NaOH having a mass concentration of 1% to obtain pretreated rice straw; second, using Cd(NO3)2·9H2O and L-cysteine ​​as sources of Cd and S to self-synthesize CdSNPs on the surface of bacteria C.kluyveri to obtain hybrid bacteria C.kluyveri-CdS; third, using the butyric acid fermentation bacterial community DCB obtained by previous breeding as an inoculum, and using the pretreated rice straw as a raw material to perform acetic acid-butyric acid fermentation; fourth, inoculating the hybrid bacteria obtained in step two into the fermentation system of step three, and using C.kluyveri-CdS as a carbon chain extension strain to perform caproic acid fermentation, thereby completing the method for enhancing the efficiency of microorganisms in producing caproic acid by utilizing rice straw based on semiconductor material modification.

[0006] Furthermore, the straw pretreatment temperature in step 1 is 50° C. and the time is 72 hours. The length of the obtained pretreated rice straw is 1 to 2 cm.

[0007] Further, the preparation method of the improved ATCC1120 for hybrid bacteria C.kluyveri-CdS culture described in step 2 is: yeast powder 2.0g / L, sodium acetate trihydrate 7.5g / L, sodium butyrate 7g / L, 2M, pH 7.0 sodium acetate buffer 1%, (NH4)2SO42.63 g / L, sodium thioglycolate 0.5g / L, biotin 5.0mcg / L, p-aminobenzoic acid 100.0mcg / L, L-cysteine ​​1g / L, 0.025% resazurin 0.4% v / v, ethanol 0.5%, MgSO4·H2O 0.25g / L, CaCl20.015 g / L, FeSO4·2H2O0.015g / L, Na2MoO4·2H2O 0.002g / L, MnSO4·2H2O 0.002g / L, prepared in 99.9% N2 environment, sterilized at 121℃ for 15min.

[0008] Furthermore, the bacterial community DCB described in step three is a medium-temperature butyric acid fermentation bacterial community obtained by early selection and breeding, and the fermentation method using the medium-temperature butyric acid fermentation bacterial community is as follows: using the fermentation broth of the anaerobic fermentation bacterial community DCB dominated by cellulose degrading bacteria, hemicellulose degrading bacteria and butyric acid fermentation bacteria as an inoculum, using PCS culture medium, adding 50 mL of fermentation broth of the anaerobic fermentation bacterial community and 20 g of pretreated rice straw per liter of PCS culture medium, the culture conditions are 37°C, 50 rpm, and fermentation is 4 days.

[0009] The preparation method of PCS medium is as follows: 20 g / L NaOH pretreated rice straw, 5 g / L peptone, 1 g / L yeast powder, 5 g / L NaCl, 2 g / L CaCO3, 0.5 g / L cysteine, nitrogen was introduced for 20 minutes to keep the medium in an anaerobic state, and sterilized at 115°C for 20 minutes.

[0010] Further, the bioaugmentation method described in step 4: hybrid bacteria C.kluyveri-CdS is inoculated into the bacterial community DCB fermentation system, the bacterial community DCB fermentation uses PCS medium, adopts a fed-batch fermentation mode, and uses pretreated rice straw as a raw material for acetic acid-butyric acid fermentation. The culture conditions are 37°C, 50rpm, and fermentation is continued until the 4th day. C.kluyveri-CdS is inoculated into the fermentation system, and OD 600 =2.0, the inoculation amount was 5%, and the system was strengthened. At the same time, 20g of pretreated rice straw, 3g of peptone and 0.5g of cysteine ​​were added on the 3rd, 5th, 7th, 9th, 11th, 14th, 17th, 19th, 21st and 23rd days of fermentation, respectively. After the addition of C.kluyveri-CdS on the 10th day, i.e. on the 14th day of fermentation, C.kluyveri-CdS was inoculated into the system again, and the OD 600 =2.0, inoculation amount is 5%, and fermentation ends after 24 days.

[0011] In step 4, after inoculating hybrid bacteria C. kluyveri-CdS to bio-enhance the DCB flora, the fermentation environment was given an alternating lighting condition of 6 h light-6 h dark at 25 W / cm 2 .

[0012] The present invention includes the following beneficial effects:

[0013] The present invention uses pretreated rice straw as raw material and PCS culture medium as the basis to prepare caproic acid. The main steps are as follows:

[0014] A. Using the previously selected mixed bacterial community DCB to carry out acetic acid-butyric acid fermentation with pretreated rice straw as substrate;

[0015] B. Using C. kluyveri as a caproic acid carbon chain extension strain;

[0016] C. Using Cd(NO3)2·9H2O and L-cysteine ​​as the source of Cd and S elements, CdS NPs were self-synthesized on the surface of bacteria C. kluyveri to construct a C. kluyveri-CdS hybrid system;

[0017] D. inoculating the hybrid bacteria obtained in step C into the fermentation system of step A for enhanced fermentation, and using C. kluyveri-CdS as a carbon chain extension strain for caproic acid fermentation;

[0018] The present invention uses pretreated rice straw as raw material, which greatly reduces the cost of raw materials; uses bacterial community DCB as inoculant for acetic acid-butyric acid fermentation, and uses photoelectrons generated by the photoelectric properties of semiconductor materials as electron donors, which can avoid the addition of electron donor ethanol and can increase the caproic acid yield, which can reach 14.6g / L. The present invention uses agricultural waste straw as raw material for biological fermentation preparation of caproic acid, which is of great significance to the recycling of resources and the development of new resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the fermentation device of Example 1; wherein 1 is a pH controller, 2 is an inoculation port, 3 is a sampling port, 4 is a water seal, 5 is a wet gas flow meter, 6 is a temperature probe, 7 is a stirring paddle, 8 is a pH probe, and 9 is a light strip.

[0020] Figure 2 This is an electron microscope image of the C.kluyveri-CdS hybrid synthesized by the present invention.

[0021] Figure 3 This is a graph showing the efficiency of enhanced caproic acid production by batch fermentation of C. kluyveri-CdS according to the present invention.

[0022] Figure 4 This is the efficiency diagram of enhanced caproic acid production by continuous fed-batch fermentation of C. kluyveri-CdS according to the present invention. DETAILED DESCRIPTION

[0023] The present invention provides a method for enhancing the efficiency of microorganisms to produce caproic acid by using rice straw based on semiconductor material modification: first, using NaOH with a mass concentration of 1% at 50°C to pretreat rice straw for 72 hours to obtain pretreated rice straw; second, using Cd(NO3)2·9H2O and L-cysteine ​​as sources of Cd and S elements respectively, culturing in a modified ATCC1120 medium for 24 hours, and C. kluyveri reaching OD 600 When the pH value is about 0.25, add filtered and sterilized Cd(NO3)2·9H2O aqueous solution to make the final concentration of Cd ions reach 0.75mM, and incubate at 170rpm for 24h, and CdS nanoparticles can be self-synthesized on the surface of C.kluyveri; third, use bacterial community DCB as inoculant, and carry out acetic acid-butyric acid fermentation with pretreated rice straw as raw material; fourth, inoculate the hybrid bacteria C.kluyveri-CdS obtained in step 2 into the fermentation system of step 3, and then use C.kluyveri as a carbon chain extension strain to carry out caproic acid fermentation, thereby completing the method of enhancing the efficiency of microorganisms to produce caproic acid using rice straw based on semiconductor material modification.

[0024] The following experiments were performed to verify the beneficial effects of the present invention:

[0025] Example 1: A method for enhancing the efficiency of microorganisms in producing caproic acid using rice straw based on semiconductor material modification

[0026] 1. Prepare the culture medium:

[0027] (1) The specific preparation method of the fermentation medium is as follows:

[0028] use Figure 1 The fermentation was carried out in the indicated fermenter with a fermentation volume of 1 L.

[0029] PCS medium: 20 g / L NaOH pretreated rice straw, 5 g / L peptone, 1 g / L yeast powder, 5 g / L NaCl, 2 g / L CaCO3, 0.5 g / L cysteine. Nitrogen was introduced for 20 minutes to keep the medium anaerobic, and sterilized at 115°C for 20 minutes. The medium preparation, inoculation, and sampling were all carried out under anaerobic conditions.

[0030] (2) Improved ATCC 1120 medium configuration:

[0031] Yeast powder 2.0 g / L, sodium acetate trihydrate 7.5 g / L, sodium butyrate 7 g / L, sodium acetate buffer (2M pH 7.0) 1%, (NH4)2SO4 2.63 g / L, sodium thioglycolate 0.5 g / L, biotin 5.0 mcg / L, p-aminobenzoic acid 100.0 mcg / L, L-cysteine ​​1 g / L, resazurin (0.025%) 0.4% (v / v), ethanol 0.5%, MgSO4·H2O 0.25 g / L, CaCl2 0.015 g / L, FeSO4·2H2O 0.015 g / L, Na2MoO4·2H2O 0.002 g / L, MnSO4·2H2O 0.002 g / L, nitrogen was introduced for 20 minutes to keep the culture medium in an anaerobic state, and sterilized at 121°C for 15 min.

[0032] 2. Synthesis of C.kluyveri-CdS hybrids:

[0033] After activation, C. kluyveri was cultured in modified ATCC 1120 medium for 24 h. 600 When the concentration of Cd(NO3)2·9H2O aqueous solution was added to reach a final concentration of 0.75 mM, the mixture was shaken at 170 rpm for 24 h. Figure 2 shown).

[0034] 3. Fermentation process:

[0035] The DCB bacteria obtained by the previous breeding was used as the inoculum, with an inoculum amount of 5% (v / v), and batch fermentation was performed using PCS medium. According to the inoculum and light conditions, the fermentation system was divided into 4 groups, of which the inoculum of the first group was the DCB bacteria, the second and third groups were the DCB bacteria + C. kluyveri-CdS bacteria, and the fourth group was the DCB bacteria + C. kluyveri. Except for the third group, all the fermentations were carried out under dark conditions. The third group was fermented under alternating lighting conditions of 6h light-6h dark (25W / cm 2 ) were fermented under the conditions of 37°C, 140 rpm, and the fermentation volume of the four groups was 50 mL, and the fermentation conditions were continuous for 7 days.

[0036] The concentration of caproic acid was determined by gas chromatography. Figure 3 As shown, after 7 days of fermentation under light conditions, the caproic acid production of the C.kluyveri-CdS enhanced system increased to 2.4 g / L, indicating that under light conditions, C.kluyveri-CdS can effectively increase the caproic acid production without adding ethanol as an electron donor.

[0037] Embodiment 2:

[0038] Different from Example 1, the fermentation process is:

[0039] (1) The fermentation volume was expanded to 1 L, and the DCB bacterial colony obtained in the early stage was used as the inoculum, with an inoculum amount of 5% (v / v). PCS medium was used for fed-batch fermentation. On the 3rd, 5th, 7th, 9th, 11th, 14th, 17th, 19th, 21st, and 23rd days of fermentation, 20 g of pretreated rice straw (dry weight), 3 g of peptone, and 0.5 g of L-cysteine ​​were added, respectively. The fermentation conditions were 37°C, pH was controlled at about 6.5, 70 rpm, and under the alternating lighting conditions of 6 h light-6 h dark (25 W / cm 2 ) for 24 days.

[0040] (2) On the 4th and 14th days of fermentation, 5% (v / v) C. kluyveri-CdS was inoculated into the bacterial DCB fermentation system, thereby completing a method for enhancing the efficiency of microorganisms in utilizing rice straw to produce caproic acid based on semiconductor material modification.

[0041] The concentration of caproic acid was determined by gas chromatography. Figure 4 As shown, the hexanoic acid yield was greatly increased from 3.0 g / L to 14.6 g / L after enhancement by C. kluyveri-CdS, and the yield increased by 3.8 times, indicating that C. kluyveri-CdS can effectively increase the hexanoic acid yield without adding ethanol as an electron donor.

[0042] In summary, the present invention uses pretreated rice straw as raw material, uses C.kluyveri-CdS hybrid system as a reinforced strain, uses the photoelectric properties of semiconductor materials, generates photoelectrons as electron donors under illumination conditions, greatly reduces the cost of raw materials, and increases the yield of caproic acid; uses bacterial community DCB as an inoculant for acetic acid-butyric acid fermentation to provide a substrate for synthesizing caproic acid, and the caproic acid yield can reach up to 14.6 g / L, achieving the purpose of reducing the additional addition of ethanol, acetic acid and butyric acid, thereby reducing production costs. The present invention uses agricultural waste straw as a raw material for the biological fermentation preparation of caproic acid, which is of great significance to the recycling of resources and the development of pollution-free new resources.

[0043] It should be further explained that the above implementation modes are only used to understand the protection scope of the present invention, and are not used to limit the protection scope of the present invention. Any obvious adjustments and modifications made to the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for enhancing the efficiency of microorganisms in producing caproic acid from rice straw based on semiconductor material modification, characterized in that The method comprises the following steps: firstly, pretreating rice straw with 1% NaOH to obtain pretreated rice straw; secondly, using Cd(NO3)2·9H2O and L-cysteine ​​as the source of Cd and S elements respectively, culturing C. kluyveri in the improved ATCC1120 medium for 24 hours until the OD 600 When the pH value is 0.25, add filtered and sterilized Cd(NO3)2·9H2O aqueous solution to make the final concentration of Cd ions reach 0.75mM, and incubate at 170rpm for 24h. CdS nanoparticles can be self-synthesized on the surface of C.kluyveri to obtain hybrid bacteria C.kluyveri-CdS; third, use the bacterial community DCB obtained in the early stage of breeding as inoculum, and use the pretreated rice straw as raw material for acetic acid-butyric acid fermentation; fourth, on the third day of DCB fermentation, add the hybrid bacteria C.kluyveri-CdS obtained in step 2 to the bacterial community DCB fermentation system for enhanced fermentation, thereby completing the method for enhancing the efficiency of microorganisms in producing caproic acid using rice straw based on semiconductor material modification.

2. The method according to claim 1, characterized in that: The straw pretreatment temperature in step 1 is 50° C. and the time is 72 hours.

3. The method according to claim 1, characterized in that: The length of the pretreated rice straw obtained in step 1 is 1 to 2 cm.

4. The method according to claim 1, characterized in that: The enhanced fermentation method of the hybrid bacteria C.kluyveri-CdS described in step 2: After successfully obtaining the C.kluyveri-CdS hybrid bacteria, collect the bacterial precipitate, transfer it to a fresh culture medium, and give it an alternating lighting condition of 6h light-6h dark.

5. The method according to claim 1, characterized in that: The preparation method of the improved ATCC1120 for culturing the hybrid bacteria C.kluyveri-CdS described in step 2 is: yeast powder 2.0 g / L, sodium acetate trihydrate 7.5 g / L, sodium butyrate 7 g / L, 2M sodium acetate buffer with pH 7.0 1%, (NH4)2SO4 2.63 g / L, sodium thioglycolate 0.5 g / L, biotin 5.0 mcg / L, p-aminobenzoic acid 100.0 mcg / L, L-cysteine ​​1 g / L, 0.025% resazurin 0.4% v / v, ethanol 0.5%, MgSO4·H2O 0.25 g / L, CaCl2 0.015 g / L, FeSO4·2H2O 0.015 g / L, Na2MoO4·2H2O 0.002 g / L, MnSO4·2H2O 0.002g / L, prepared in 99.9% N2 environment, sterilized at 121℃ for 15min.

6. The method according to claim 1, characterized in that: The DCB flora described in step three is a medium-temperature butyric acid fermentation flora obtained by early breeding. The fermentation method using the medium-temperature butyric acid fermentation flora is as follows: using the fermentation broth of the anaerobic fermentation flora DCB dominated by cellulose-degrading bacteria, hemicellulose-degrading bacteria and butyric acid fermentation bacteria as an inoculum, using PCS culture medium, adding 50 mL of fermentation broth of the anaerobic fermentation flora and 20 g of pretreated rice straw per liter of PCS culture medium, the culture conditions are 37°C, 50 rpm, and the fermentation is 4 days.

7. The method according to claim 6, characterized in that: The preparation method of PCS medium is as follows: 20 g / L NaOH pretreated rice straw, 5 g / L peptone, 1 g / L yeast powder, 5 g / L NaCl, 2 g / L CaCO3, 0.5 g / L cysteine, nitrogen is introduced for 20 minutes to keep the medium in an anaerobic state, and sterilized at 115°C for 20 minutes.

8. The method of claim 1, wherein the semiconductor material is used to modify microorganisms to enhance the efficiency of producing caproic acid from rice straw, The bioaugmentation method described in step 4 is as follows: hybrid bacteria C.kluyveri-CdS is inoculated into the bacterial community DCB fermentation system, the bacterial community DCB fermentation uses PCS medium, adopts a fed-batch fermentation mode, and uses pretreated rice straw as a raw material for acetic acid-butyric acid fermentation. The culture conditions are 37°C, 50rpm, and fermentation is continued until the 4th day. C.kluyveri-CdS is inoculated into the fermentation system, and OD 600 =2.0, the inoculation amount was 5%, and the system was strengthened. At the same time, 20g of pretreated rice straw, 3g of peptone and 0.5g of cysteine ​​were added on the 3rd, 5th, 7th, 9th, 11th, 14th, 17th, 19th, 21st and 23rd days of fermentation, respectively. After the addition of C.kluyveri-CdS on the 10th day, i.e. on the 14th day of fermentation, C.kluyveri-CdS was inoculated into the system again, and the OD 600 =2.0, inoculation amount is 5%, and fermentation ends after 24 days.

9. The method according to claim 1, characterized in that: Step 4 also includes the following steps: after inoculating the hybrid bacteria C. kluyveri-CdS to bio-enhance the DCB flora, the fermentation environment is given an alternating lighting condition of 6 h light-6 h dark at 25 W / cm 2 .

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