CN113247946A - Self-contained nano biocatalyst, preparation method thereof and application thereof in butanol production - Google Patents
Self-contained nano biocatalyst, preparation method thereof and application thereof in butanol production Download PDFInfo
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- CN113247946A CN113247946A CN202110439068.XA CN202110439068A CN113247946A CN 113247946 A CN113247946 A CN 113247946A CN 202110439068 A CN202110439068 A CN 202110439068A CN 113247946 A CN113247946 A CN 113247946A
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- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
The invention discloses an independently-loaded nano biocatalyst, a preparation method thereof and application thereof in butanol production, belonging to the field of microbial preparation and application of nano materials. The invention solves the problems of slow butanol fermentation efficiency and low yield in the existing biological butanol preparation process. The patent provides an independently-installed nano biocatalyst, the catalyst is obtained by modifying butanol fermentation strains by using a nano cadmium sulfide material, the excellent characteristics of strong specificity and less pollution of the biological butanol fermentation strains at room temperature are kept, and meanwhile, the high efficiency of the nano cadmium sulfide photocatalyst is utilized, so that the butanol fermentation rate and the yield are greatly improved, and a new way is provided for the high-efficiency preparation of the biological butanol.
Description
Technical Field
The invention relates to an independently-loaded nano biocatalyst, a preparation method thereof and application thereof in butanol production, belonging to the field of microbial preparation and application of nano materials.
Background
Catalysis is an important phenomenon ubiquitous in nature, and its role extends almost throughout the entire field of chemical reactions. The practice of chemical processing by catalysis has a long history. In the 19-20 th century, the material processing industry taking nonrenewable fossil resources as an economic basis has gained brilliant achievements, and in the 21 st century, in the face of the serious situation that the fossil resources are continuously exhausted and the environmental pollution is increasingly aggravated, human beings face unprecedented crises of survival and development. The traditional substance processing industry has to revolutionarily change to the biological processing industry which takes biological renewable resources as raw materials, is environment-friendly and efficient in process, and has the core technology of biological catalysis and biological conversion. Therefore, the sustainable development of human society urgently needs biocatalysis and biotransformation technology.
Industrial biotechnology is emerging as a future, and industrial biocatalysts thereof are generally carried out in a closed system, can conveniently eliminate hidden troubles after use, and have the characteristic of high biosafety. However, in the biological catalysis and the biological conversion, several key problems of limited biological catalyst resources, narrow application range, low efficiency and the like still exist.
As an excellent bio-liquid fuel, bio-butanol production technology has received extensive attention and research. However, the slow fermentation rate of butanol, a large amount of fermentation byproducts and the like are insufficient, and the popularization and application of the biological butanol technology are severely restricted. Therefore, it is necessary to provide a more economical and efficient bio-butanol production technology.
Disclosure of Invention
The invention aims to solve the problems of slow butanol fermentation efficiency and low yield in the traditional biological butanol preparation process.
The invention also aims to provide a method for biologically synthesizing the nano cadmium sulfide semiconductor material, which has the advantages of mild conditions, high dispersity, strong photocatalytic effect and the like compared with chemical synthesis.
The technical scheme of the invention is as follows:
a self-assembled nano-biocatalyst consists of thalli and nano-particles on the surfaces of the thalli, wherein the thalli is clostridium beijerinckii, and the nano-particles are nano cadmium sulfide.
The self-assembled nano biocatalyst is obtained by a biomimetic mineralization method of clostridium beijerinckii mixed nano cadmium sulfide semiconductor material, and the method comprises the following steps: clostridium beijerinckii and Cd (NO)3)2Co-inoculating in a P2 culture medium for culture, collecting precipitates after the culture is finished, and washing to obtain the self-contained nano biocatalyst.
Further limiting, the specific operation process for preparing the self-contained nano biocatalyst is as follows: inoculating Clostridium beijerinckii into P2 culture medium at a volume ratio of 1:20, and inoculating Cd (NO) into P2 culture medium at the beginning of culture3)2Then culturing for 60h under the conditions of 140rpm and 37 ℃, collecting precipitates after culturing, and washing to obtain the self-contained nano biocatalyst.
Further defined, Cd (NO)3)2The concentration of the culture medium with the inoculation amount P2 is 0.1g/L, 0.2g/L or 0.3 g/L.
Further limited, Cd (NO)3)2The inoculation mode is as follows: mixing Cd (NO) with anaerobic water3)2·4H2O is prepared into mother liquor with the concentration of 5g/L, and then the mother liquor is proportionally inoculated into P2 culture medium.
Further limiting, the P2 culture medium is yeast extract powder 3g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, cysteine 0.5g/L, magnesium sulfate 0.2g/L, P-aminobenzoic acid 0.001g/L, thiamine 0.001g/L, biotin 0.0001g/L, manganese sulfate 0.01g/L, ferrous sulfate 0.01g/L, sodium chloride 0.01g/L, and glucose 10 g/L.
Further limiting, the preparation of P2 culture medium requires anaerobic operation, boiling appropriate amount of deionized water, introducing nitrogen gas for 10-20min, dissolving the above medicines in water, packaging, and sterilizing at 115 deg.C for 20 min.
Further defined, Clostridium beijerinckii NCIMB 8052, obtained from the China center for microbiological culture Collection center (CGMCC).
More particularly, the clostridium beijerinckii needs to be activated and cultured by using a P2 culture medium before being used.
The method for producing butanol by applying the self-contained nano biocatalyst prepared by the method comprises the following steps: inoculating the self-assembled nano biocatalyst into a P2 culture medium, culturing for 24h under illumination, and performing butanol fermentation by using the self-assembled nano biocatalyst.
Further limiting, the culture conditions under light are as follows: the light intensity is 2000W/m2The temperature was 37 ℃ and the rotational speed was 140 rpm.
Compared with chemical synthesis, the self-contained nano biocatalyst prepared by the method for producing the nano cadmium sulfide semiconductor material has the advantages of mild conditions, high dispersibility, strong photocatalytic effect and the like.
The invention has the following beneficial effects:
(1) the invention utilizes clostridium beijerinckii as a biological mineralizer to synthesize the cadmium sulfide semiconductor nano material with high-efficiency photocatalysis capacity on the cell membrane of the clostridium beijerinckii, thereby synthesizing the self-assembled nano biocatalyst. Compared with the butanol fermentation effect of pure bacteria, the self-assembled nano biocatalyst keeps the butanol production capacity of butanol fermentation bacteria, is coupled with excellent semiconductor nanoparticle catalysts, and has more excellent butanol fermentation capacity under the illumination condition.
(2) The method for synthesizing nano cadmium sulfide semiconductor material is characterized by adding proper quantity of Cd (NO) into P2 culture medium3)2Cadmium is a main pollutant for increasing ecological risks, and the treatment methods of cadmium are mainly precipitation, adsorption, ion exchange and the like. Wherein precipitation is a more efficient process. The method of biomineralization can effectively precipitate cadmium in the environment so as to reduce the toxicity of the cadmium.
(3) The method has the advantages of cheap and easily-obtained raw materials, few synthesis steps, high reaction controllability, mild synthesis conditions, simple and feasible separation method and lower production and use costs.
Drawings
FIG. 1 shows different Cd (NO)3)2Biomass comparison curve chart of the co-cultured thalli under the addition amount;
FIG. 2 is a transmission electron microscope result chart of the Clostridium beijerinckii strain of example 1;
FIG. 3 is a transmission electron microscope result chart of the self-contained nano biocatalyst of example 2;
FIG. 4 is a transmission electron microscope result chart of the self-contained nano biocatalyst of example 3;
FIG. 5 is a transmission electron microscope result chart of the self-contained nano biocatalyst of example 4;
FIG. 6 is an XRD analysis of the self-contained nano-biocatalyst of example 2;
FIG. 7 is an XRD analysis of the self-contained nano-biocatalyst of example 3;
FIG. 8 is an XRD analysis of the self-contained nano-biocatalyst of example 4;
FIG. 9 is a graph comparing the yields of butanol produced by catalytic fermentation with self-contained nano biocatalysts obtained in examples 1-4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents, methods and apparatus used, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.
Example 1: blank group
(1) Obtaining the strains of clostridium beijerinckii: inoculating the preserved clostridium beijerinckii bacterial liquid into a P2 culture medium for activated culture, then inoculating the bacterial liquid into a fresh P2 culture medium according to the proportion of 1:20, culturing the bacterial liquid for 60 hours under the conditions of 140rpm and 37 ℃ to obtain the clostridium beijerinckii bacterial liquid, and measuring the biomass of the obtained clostridium beijerinckii bacterial liquid, wherein the test result is shown in figure 1. As can be seen from FIG. 1, NO Cd (NO) is present3)2In the case of addition, OD of Clostridium beijerinckii bacterial liquid after 60 hours of culture600Reaching 2.23 +/-0.11, and showing that the strain has good growth state.
Transmitting electrons to the obtained Clostridium beijerinckii thallusThe scanning electron microscope test shows that the test result is shown in FIG. 2. As is clear from FIG. 2, the surface of the Clostridium beijerinckii cells of this example did not produce any nanomaterial, indicating that there was NO Cd (NO)3)2The cadmium sulfide nano material is not generated under the condition of adding.
(2) Butanol fermentation: centrifuging to collect thallus, inoculating the obtained thallus of Clostridium beijerinckii into fresh P2 culture medium, and culturing at 2000W/m2Culturing at 37 deg.C and 140rpm for 24 h. And performing butanol fermentation test, wherein the test result is shown in figure 9, and the butanol yield can reach 1.66 +/-0.17 g/L after 24h of fermentation.
Example 2: adding 0.1g/L Cd (NO)3)2
(1) Obtaining the self-contained nano biocatalyst: inoculating the preserved Clostridium beijerinckii bacterial liquid into a P2 culture medium for activation culture, then inoculating the bacterial liquid into a fresh P2 culture medium according to the proportion of 1:20, and simultaneously inoculating 0.1g/L Cd (NO) into a P2 culture medium3)2(the access mode is that the Cd (NO) is mixed with anaerobic water3)2·4H2O is prepared into mother liquor with the concentration of 5g/L, then the mother liquor is proportionally inoculated into P2 culture medium), and then the mother liquor is cultured for 60 hours according to the conditions of 140rpm and 37 ℃.
The biomass of the obtained clostridium beijerinckii bacterial liquid was measured, and the test results are shown in fig. 1. As can be seen from FIG. 1, Cd (NO) at 0.1g/L3)2In the case of addition, OD of Clostridium beijerinckii bacterial liquid after 60 hours of culture600Reaching 2.91 +/-0.02, and showing that the growth state of the strain is promoted by low-concentration cadmium ions.
The transmission electron scanning electron microscope test was performed on the obtained clostridium beijerinckii bacterial liquid, and the test result is shown in fig. 3. As can be seen from FIG. 3, the surface of the Clostridium beijerinckii strain of this example has nanoparticles, but the extracellular portion has no nanoparticles, indicating that the self-contained nano-biocatalyst was successfully prepared.
The synthesized nanomaterial was collected and subjected to XRD testing, the results of which are shown in fig. 6. As can be seen from fig. 6, the nanoparticles on the surface of the clostridium beijerinckii bacteria exist as crystals, and the absorption peak of the nanoparticles accords with the absorption peak of the cadmium sulfide nanomaterial, which proves that the cadmium sulfide nanomaterial is successfully prepared.
The reason is that the clostridium beijerinckii is a butanol fermentation strain, a large amount of components such as protein, polypeptide, carbohydrate, lipid and the like exist on the cell surface of the clostridium beijerinckii, and a plurality of groups exist in the biological components, so that cadmium ions can be gathered on the cell surface. Clostridium beijerinckii degrades the cysteine component of the medium during growth and metabolism, thereby generating and releasing H2S gas, H2The S gas reacts with cadmium ions on the cell surface to generate the nano cadmium sulfide material.
(2) Butanol fermentation: centrifugally collecting the self-contained nano biocatalyst, and re-inoculating the self-contained nano biocatalyst into a fresh P2 culture medium at 2000W/m2Culturing at 37 deg.C and 140rpm for 24 h. And performing butanol fermentation test, wherein the test result is shown in figure 9, the butanol yield can reach 2.18 +/-0.21 g/L after 24h of fermentation, and the butanol yield is greatly improved.
The method is characterized in that the organic components on the surface of the bacteria are regulated, so that the nano cadmium sulfide material with small volume, large specific surface area, high dispersibility and high stability is formed on the surface of a cell membrane, and the electron transfer is relatively quick under the illumination condition. Under illumination, the nano cadmium sulfide material has the function of plant chlorophyll, and provides possibility for efficient synthesis of a reducing product butanol.
Example 3: adding 0.2g/L Cd (NO)3)2
(1) Obtaining the self-contained nano biocatalyst: inoculating the preserved Clostridium beijerinckii bacterial liquid into a P2 culture medium for activation culture, then inoculating the bacterial liquid into a fresh P2 culture medium according to the proportion of 1:20, and simultaneously inoculating 0.2g/L Cd (NO) into a P2 culture medium3)2The cells were cultured at 37 ℃ and 140rpm for 60 hours.
The biomass of the obtained clostridium beijerinckii bacterial liquid was measured, and the test results are shown in fig. 1. As can be seen from FIG. 1, Cd (NO) at 0.2g/L3)2In the case of addition, OD of Clostridium beijerinckii bacterial liquid after 60 hours of culture600Reaching 1.12 +/-0.05, and showing that the growth state of the strain is inhibited by cadmium ions.
The transmission electron scanning electron microscope test was performed on the obtained clostridium beijerinckii bacterial liquid, and the test result is shown in fig. 4. As can be seen from FIG. 4, the surface of the Clostridium beijerinckii strain of this example has nanoparticles, but the extracellular portion has no nanoparticles, indicating that the self-contained nano-biocatalyst was successfully prepared.
The synthesized nanomaterial was collected and subjected to XRD testing, the results of which are shown in fig. 7. As can be seen from fig. 7, the nanoparticles on the surface of the clostridium beijerinckii bacteria exist as crystals, and the absorption peak of the nanoparticles accords with the absorption peak of the cadmium sulfide nanomaterial, which proves that the cadmium sulfide nanomaterial is successfully prepared.
(2) Butanol fermentation: centrifugally collecting the self-contained nano biocatalyst, and re-inoculating the self-contained nano biocatalyst into a fresh P2 culture medium at 2000W/m2Culturing at 37 deg.C and 140rpm for 24 h. And performing a butanol fermentation test, wherein the test result shows that the butanol yield is only 1.18 +/-0.18 g/L after 24h of fermentation as shown in Table 9.
Example 4: adding 0.3g/L Cd (NO)3)2
(1) Obtaining the self-contained nano biocatalyst: inoculating the preserved Clostridium beijerinckii bacterial liquid into a P2 culture medium for activation culture, then inoculating the bacterial liquid into a fresh P2 culture medium according to the proportion of 1:20, and simultaneously inoculating 0.3g/L Cd (NO) into a P2 culture medium3)2The cells were cultured at 37 ℃ and 140rpm for 60 hours.
The biomass of the obtained clostridium beijerinckii bacterial liquid was measured, and the test results are shown in fig. 1. As can be seen from FIG. 1, Cd (NO) at 0.3g/L3)2In the case of addition, OD of Clostridium beijerinckii bacterial liquid after 60 hours of culture600Only 1.12 +/-0.08 shows that the growth state of the strain is inhibited by high-concentration cadmium ions.
The transmission electron scanning electron microscope test was performed on the obtained clostridium beijerinckii bacterial liquid, and the test result is shown in fig. 5. As can be seen from FIG. 5, the surface of the Clostridium beijerinckii strain of this example has nanoparticles, but the extracellular portion has no nanoparticles, indicating that the self-contained nano-biocatalyst was successfully prepared.
The synthesized nanomaterial was collected and subjected to XRD testing, the results of which are shown in fig. 8. As can be seen from fig. 8, the nanoparticles on the surface of the clostridium beijerinckii bacteria exist as crystals, and the absorption peak of the nanoparticles accords with the absorption peak of the cadmium sulfide nanomaterial, which proves that the cadmium sulfide nanomaterial is successfully prepared.
(2) Butanol fermentation: centrifugally collecting the self-contained nano biocatalyst, and re-inoculating the self-contained nano biocatalyst into a fresh P2 culture medium at 2000W/m2Culturing at 37 deg.C and 140rpm for 24 h. And performing butanol fermentation test, wherein the test result is shown in Table 9, and the butanol yield is only 0.53 +/-0.04 g/L after 24h of fermentation.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The self-assembled nano biocatalyst is characterized by comprising thalli and nano particles on the surfaces of the thalli, wherein the thalli is clostridium beijerinckii, and the nano particles are nano cadmium sulfide.
2. A method for preparing the self-assembled nano biocatalyst as claimed in claim 1, wherein the method comprises: clostridium beijerinckii and Cd (NO)3)2Co-inoculating in a P2 culture medium for culture, collecting precipitates after the culture is finished, and washing to obtain the self-contained nano biocatalyst.
3. The preparation method of the self-assembled nano biocatalyst according to claim 2, characterized by comprising the following specific procedures: inoculating Clostridium beijerinckii into P2 culture medium at a volume ratio of 1:20, and inoculating Cd (NO) into P2 culture medium at the beginning of culture3)2Then culturing for 60h under the conditions of 140rpm and 37 ℃, collecting precipitates after culturing, and washing to obtain the self-contained nano biocatalyst.
4. The method for preparing the self-assembled nano-biocatalyst of claim 2 or 3, wherein the Cd (NO) is3)2Access volumeThe concentration of the P2 medium is 0.1g/L, 0.2g/L or 0.3 g/L.
5. The method for preparing the self-assembled nano biocatalyst as claimed in claim 2, wherein the Cd (NO) is3)2The inoculation mode is as follows: mixing Cd (NO) with anaerobic water3)2·4H2O is prepared into mother liquor with the concentration of 5g/L, and then the mother liquor is proportionally inoculated into P2 culture medium.
6. The method for preparing the self-assembled nano biocatalyst of claim 2, wherein the P2 culture medium is yeast extract powder 3g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, cysteine 0.5g/L, magnesium sulfate 0.2g/L, P-aminobenzoic acid 0.001g/L, thiamine 0.001g/L, biotin 0.0001g/L, manganese sulfate 0.01g/L, ferrous sulfate 0.01g/L, sodium chloride 0.01g/L, glucose 10 g/L.
7. The method for preparing a self-assembled nano-biocatalyst as claimed in claim 2, wherein said Clostridium beijerinckii NCIMB 8052 is Clostridium stickeriun beijerinckii.
8. The method for producing butanol using the self-assembled nano biocatalyst of claim 1, wherein the method comprises: inoculating the self-assembled nano biocatalyst into a P2 culture medium, culturing for 24h under illumination, and performing butanol fermentation by using the self-assembled nano biocatalyst.
9. The method for producing butanol using self-assembled nanobiocysts according to claim 8, wherein the culture conditions under light are: the light intensity is 2000W/m2The temperature was 37 ℃ and the rotational speed was 140 rpm.
10. The method for preparing the self-assembled nano biocatalyst according to claim 2 is used for producing the nano cadmium sulfide semiconductor material.
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