Method for biosynthesis of gold nanoparticles and application thereof

Abstract

The application relates to a biosynthesis method of gold nanoparticles, which comprises taking gold ions as gold nanoparticle precursors, taking microorganisms as biocatalysts to carry out a reduction reaction of the gold ions to obtain the gold nanoparticles; the reaction condition is mild, the method is environment-friendly, simple and easy to operate, and the synthesized gold nanoparticles have high biocompatibility and catalytic activity. The gold nanoparticles prepared by the method can be applied to the fermentation hydrogen production of hydrogen production bacteria.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterial preparation and biological hydrogen production, and relates to a biosynthesis method of gold nanoparticles and application thereof. BACKGROUND

[0002] Due to high surface area and surface plasmon resonance effect (LSPR), gold nanoparticles are considered as a promising catalyst. In addition, based on the unique properties of gold nanoparticles such as chemical stability, high biocompatibility, etc., gold nanoparticles are also widely used in biomedical fields such as drug delivery, photothermal therapy, cosmetics, biosensors, etc. However, the current gold nanoparticle synthesis method mainly adopts various physical and chemical methods. The chemical method uses various toxic reagents in the synthesis process or cannot effectively guarantee the safety problem in the later application, so that the chemical method is not green and environmentally friendly, which limits the application of gold nanoparticles in various biological fields. Therefore, it is necessary to develop a synthesis method of gold nanoparticles with low toxicity, high biocompatibility and environmental protection.

[0003] The biosynthesis of gold nanoparticles is achieved by using plant extract or microorganism to reduce gold salt ion solution. In this process, the plant extract or microorganism acts as a reducing agent, a stabilizer and an end-capping agent at the same time, and no additional stabilizer needs to be added. The biosynthesis method of gold nanoparticles has become a promising method for preparing gold nanoparticles based on its advantages such as environmental protection, non-pathogenicity, economy, mild synthesis conditions, etc.

[0004] However, the method for biosynthesis of gold nanoparticles is not mature at present. The time, temperature and other factors of the biosynthesis method of gold nanoparticles will affect the synthesis of gold nanoparticles, and the size and shape of the biosynthesis of gold nanoparticles still need to be further optimized (Ahmed S, Ikram S. Biosynthesis of gold nanoparticles: a green approach [J]. Journal of Photochemistry and Photobiology B: Biology, 2016, 161: 141-153). Therefore, it is necessary to develop a rapid and simple biosynthesis method of gold nanoparticles. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a biosynthesis method of gold nanoparticles in view of the problems existing in the prior art. The biosynthesis method has the advantages of mild reaction conditions, environmental friendliness, simple and easy operation, etc. The synthesized gold nanoparticles have high biocompatibility and catalytic activity. The mixed system composed of the gold nanoparticles prepared by the method and hydrogen-producing bacteria is subjected to anaerobic culture and photo-fermentation to produce hydrogen, and the hydrogen production of the mixed system is significantly higher than that of the naked Clostridium butyricum subjected to photo / dark fermentation.

[0006] To this end, the present application provides a biosynthesis method of gold nanoparticles, which comprises using gold ions as precursors of gold nanoparticles and using microorganisms as biocatalysts to reduce the gold ions to obtain gold nanoparticles.

[0007] According to the present application, the biosynthesis method comprises a step of preparing gold nanoparticles, which comprises reducing the gold ions after mixing the gold ions as precursors of gold nanoparticles with biocatalysts and a reducing reaction solvent to obtain a bacterial solution containing gold nanoparticles; in the bacterial solution containing gold nanoparticles, the gold nanoparticles exist in the form of free gold nanoparticles and / or in the form of gold nanoparticle-biocatalyst hybrid.

[0008] In the present application, the gold ions are gold acid and / or gold salt capable of dissociating gold ions in water, which comprises one or more of chloroauric acid, sodium gold thiosulfate and gold chloride.

[0009] In some embodiments of the present application, the working concentration of the gold ions is 0.25-2 mmol / L, more preferably 0.5-1 mmol / L.

[0010] In the present application, the biocatalysts comprise one or more of Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Enterobacter aerogenes and Escherichia coli.

[0011] In some embodiments of the present application, the working concentration of the biocatalysts is OD600 value of 0.1-5.

[0012] Preferably, the reducing reaction solvent comprises one or more of bacterial culture medium, physiological saline, phosphate buffer and water, more preferably bacterial culture medium or physiological saline; further more preferably, the bacterial culture medium comprises reinforced Clostridium culture medium and LB culture medium for Enterobacter.

[0013] In some embodiments of the present application, the temperature of the reducing reaction is 25-40℃, preferably 37-40℃.

[0014] In some embodiments of the present application, the time of the reducing reaction is 6-48h, preferably 12-36h, further preferably 24-36h.

[0015] In some embodiments of the present application, the rotating speed of the reducing reaction is 0-180 rpm, preferably 120-180 rpm.

[0016] According to the present application, the biosynthesis method further comprises a step of separating and purifying the gold nanoparticles and / or the gold nanoparticle-biocatalyst hybrid, and the gold nanoparticles and / or the gold nanoparticle-biocatalyst hybrid in the bacterial solution containing the gold nanoparticles are separated and purified to obtain gold nanoparticle products and / or gold nanoparticle-biocatalyst hybrid products.

[0017] The present application provides a gold nanoparticle prepared by the biosynthesis method according to the first aspect of the present application.

[0018] The present application provides a gold nanoparticle-biocatalyst hybrid prepared by the biosynthesis method according to the first aspect of the present application, which is composed of gold nanoparticles uniformly attached to the whole biocatalyst.

[0019] In the present application, the biocatalyst comprises one or more of Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Enterobacter aerogenes and Escherichia coli, and is preferably Clostridium butyricum.

[0020] The present application provides a use of the gold nanoparticle prepared by the biosynthesis method according to the first aspect of the present application in biological hydrogen production.

[0021] According to the present application, the use comprises a step of preparing a hydrogen production culture system containing the gold nanoparticles, which comprises mixing the gold nanoparticles and the hydrogen production bacteria in a hydrogen production culture solution to form the hydrogen production culture system containing the gold nanoparticles.

[0022] Preferably, the hydrogen production bacteria are Clostridium butyricum, and correspondingly, the hydrogen production culture solution is a Clostridium butyricum hydrogen production culture solution.

[0023] In some preferred embodiments of the present application, the step of preparing the hydrogen production culture system containing the gold nanoparticles comprises mixing the bacterial solution containing the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application in a hydrogen production culture solution to form the hydrogen production culture system containing the gold nanoparticles, wherein the gold nanoparticles in the bacterial solution containing the gold nanoparticles exist in the form of free gold nanoparticles and / or in the form of gold nanoparticle-biocatalyst hybrid, and preferably in the form of gold nanoparticle-biocatalyst hybrid.

[0024] Preferably, the inoculation amount of the bacterial solution containing the gold nanoparticles in the hydrogen production culture system is 5%-20% of the total volume of the fermentation system, and preferably 10%-20%.

[0025] According to the present application, the use further comprises a step of biological hydrogen production culture, which comprises biological hydrogen production culture of the hydrogen production culture system containing the gold nanoparticles.

[0026] Preferably, the light source of the biological hydrogen production culture comprises one or more of LED visible light, natural light and xenon lamp.

[0027] In some embodiments of the present application, the light intensity of the biological hydrogen production culture is 0-5 mW / cm 2 , preferably 2.5 mW / cm 2 .

[0028] The present application has the advantages that the shape and size of the gold nanoparticles prepared by the method of the present application can be regulated by the reaction time, and the gold nanoparticles can be directly applied to the biological catalytic reaction of the gold nanoparticle synthesis bacteria (biological catalyst for synthesizing gold nanoparticles), thereby significantly improving the production capacity of the target product of the gold nanoparticle synthesis bacteria (biological catalyst for synthesizing gold nanoparticles). For example, when the strict anaerobic hydrogen-producing bacterium Clostridium butyricum is used as the biological catalyst for synthesizing gold nanoparticles by the biological method, the gold nanoparticles with uniform particle size and regular shape can be synthesized (0.5 mM chloroauric acid in a physiological saline system), and the gold nanoparticles synthesized by Clostridium butyricum can significantly promote the anaerobic fermentation hydrogen production capacity of Clostridium butyricum under light. The gold nanoparticle synthesis method of the present application has the advantages of simple operation and environmental friendliness, and the synthesized gold nanoparticles have the advantages of high biocompatibility and catalytic activity. At the same time, the gold nanoparticle-biological catalyst (for example, Clostridium butyricum) hybrid prepared by the method of the present application is inoculated in the hydrogen production fermentation system of Clostridium butyricum for photofermentation, and the hydrogen production capacity of the hybrid system is significantly higher than that of the naked free Clostridium butyricum. BRIEF DESCRIPTION OF DRAWINGS

[0029] The present application will be further described in detail below in combination with the accompanying drawings:

[0030] Figure 1Transmission electron microscopy images of gold nanoparticles prepared for the experimental group of Example 1 of the present application and Clostridium butyricum: (a) bright field transmission electron microscopy image of Clostridium butyricum cultured in reinforced Clostridium culture medium for 24 h, (b) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under bright field transmission electron microscopy after Clostridium butyricum reduced 0.5 mM chloroauric acid in reinforced Clostridium culture medium for 24 h, (c) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under dark field transmission electron microscopy after Clostridium butyricum reduced 0.5 mM chloroauric acid in reinforced Clostridium culture medium for 24 h, (d) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under bright field transmission electron microscopy after Clostridium butyricum reduced 2 mM chloroauric acid in reinforced Clostridium culture medium for 24 h, (e) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under bright field transmission electron microscopy after Clostridium butyricum reduced 0.5 mM chloroauric acid in physiological saline for 24 h, (f) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under dark field transmission electron microscopy after Clostridium butyricum reduced 0.5 mM chloroauric acid in physiological saline for 24 h, (g) morphology of the hybrid of Clostridium butyricum and gold nanoparticles under bright field transmission electron microscopy after Clostridium butyricum reduced 2 mM chloroauric acid in physiological saline for 24 h.

[0031] Figure 2 Graph showing the reduction efficiency of chloroauric acid by different microorganisms in Example 2 of the present application

[0032] Figure 3 Graph showing the reduction efficiency of chloroauric acid by Clostridium butyricum in Example 3 of the present application.

[0033] Figure 4 Graph showing the reduction efficiency of 0.5 mM chloroauric acid by Clostridium butyricum in physiological saline and reinforced Clostridium culture medium in Example 4 of the present application.

[0034] Figure 5 Bar graph showing the cumulative hydrogen production of the hybrid of gold nanoparticles and Clostridium butyricum after dark / light fermentation for 144 h in Example 5 of the present application. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the present application will be described in detail below with reference to the accompanying drawings. However, before describing the present application in detail, it should be understood that the present application is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0036] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0037] I. Terminology

[0038] The term "water" as used herein, unless otherwise specified or limited, refers to deionized water, distilled water or ultrapure water.

[0039] The term "working concentration" as used herein refers to the concentration of the reactant in the reaction system.

[0040] The term "fermentation system" as used herein refers to the improved Clostridium culture medium / base for hydrogen production by fermentation.

[0041] II. Embodiments

[0042] As described above, the current biological method for synthesizing gold nanoparticles is not mature, and factors such as time and temperature will affect the synthesis of gold nanoparticles, and the size and shape of the synthesized gold nanoparticles still need to be further optimized. In view of this, the inventors have carried out a lot of research on the biological synthesis of gold nanoparticles.

[0043] The inventors have found that using chloroauric acid as a precursor of gold nanoparticles and using Clostridium butyricum to reduce chloroauric acid to prepare gold nanoparticles is simple, fast, and has mild reaction conditions, and the synthesized gold nanoparticles have high biocompatibility and catalytic activity.

[0044] The inventors have further found that using gold ions as a precursor of gold nanoparticles and using microorganisms as a biological catalyst for reduction of gold ions, a large number of gold nanoparticles exist in the form of gold nanoparticle-biological catalyst hybrid in the bacterial solution containing gold nanoparticles obtained within a certain reaction time during the reduction reaction; and the inventors have found that the hydrogen production capacity of the hybrid system is significantly higher than that of naked free Clostridium butyricum when the bacterial solution containing gold nanoparticle-biological catalyst (e.g., Clostridium butyricum) hybrid is inoculated into a Clostridium butyricum hydrogen production fermentation system for photofermentation.

[0045] In addition, the inventors have noticed that biological hydrogen is a clean and green renewable energy, but low hydrogen yield has always restricted the industrial production of biological hydrogen. Therefore, the inventors have researched and tried to use the synthesized gold nanoparticles to affect the catalytic activity of the gold nanoparticle synthesis bacteria to establish an efficient gold nanoparticle-biological hybrid efficient photo-biological catalytic hydrogen production system for hydrogen production, and the hydrogen production is significantly higher than that of naked Clostridium butyricum system. Based on the above, the present application is obtained.

[0046] Therefore, the biological synthesis method of gold nanoparticles according to the first aspect of the present application mainly uses gold ions as a precursor of gold nanoparticles and uses microorganisms as a biological catalyst for reduction of gold ions to obtain gold nanoparticles.

[0047] Specifically, the biosynthesis method of the gold nanoparticles in the present application comprises the step of preparing the gold nanoparticles, which comprises reducing the gold ions after mixing the gold ions as the precursors of the gold nanoparticles with the biological catalyst and the reducing reaction solvent, and obtaining the bacterial solution containing the gold nanoparticles; in the bacterial solution containing the gold nanoparticles, the gold nanoparticles exist in the form of free gold nanoparticles and / or gold nanoparticle-biological catalyst hybrid.

[0048] In some embodiments of the present application, the bacterial solution containing the gold nanoparticles contains the gold nanoparticle-biological catalyst hybrid and the biological catalyst.

[0049] In some other embodiments of the present application, the bacterial solution containing the gold nanoparticles contains the gold nanoparticle-biological catalyst hybrid, the biological catalyst and a small amount of free gold nanoparticles.

[0050] In the present application, the gold ions are the gold acid and / or gold salt capable of dissociating gold ions in water, which can be understood as the gold ions exist in the form of the gold acid and / or gold salt capable of dissociating gold ions in water, which includes one or more of chloroauric acid (HAuCl4), sodium gold thiosulfate and gold chloride, and is preferably gold chloric acid.

[0051] In some embodiments of the present application, the working concentration of the gold ions is 0.25-2 mmol / L, and more preferably 0.5-1 mmol / L.

[0052] The research results show that in the process of preparing the gold nanoparticles, any one of Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Enterobacter aerogenes and Escherichia coli can be used as the biological catalyst to reduce the gold ions into the gold nanoparticles. Moreover, since the subsequent biological hydrogen production process uses Clostridium butyricum with high biological hydrogen synthesis activity as the hydrogen-producing bacteria, in order to directly use the bacterial solution containing the gold nanoparticles for hydrogen production and simplify the process, it is preferred that Clostridium butyricum is used as the biological catalyst in the process of preparing the gold nanoparticles.

[0053] In some embodiments of the present application, the working concentration of the biological catalyst is OD600 value of 0.1-5.

[0054] In the present application, the reducing reaction solvent includes one or more of the bacterial culture medium, physiological saline, phosphate buffer and water, and is more preferably the bacterial culture medium or physiological saline; further more preferably, the bacterial culture medium includes the reinforced Clostridium culture medium and the LB culture medium for culturing Enterobacter.

[0055] The inventors have found that the use of bacterial culture medium as the reducing reaction solvent is also beneficial to the reducing reaction, although it is mainly considered to be beneficial to maintaining the activity of the bacteria. Surprisingly, the reduction reaction of gold ions using gold ions as precursors of gold nanoparticles and microorganisms as biocatalysts can be well carried out in physiological saline, phosphate buffer, and even in water, especially when physiological saline is used as the reducing reaction solvent, the reduction reaction efficiency is greater than when phosphate buffer and water are used as the reducing reaction solvent, and is even significantly higher than when bacterial culture medium is used as the reducing reaction solvent.

[0056] Further research has found that the reduction reaction of gold ions using the biosynthesis method of the present application can be carried out under very mild conditions.

[0057] For example, in some embodiments of the present application, the temperature of the reduction reaction is 25-40°C, preferably 37-40°C, and more preferably 37°C.

[0058] For another example, in some embodiments of the present application, the time of the reduction reaction is 6-48h, preferably 12-36h, more preferably 24-36h, and further more preferably 24h.

[0059] For another example, in some embodiments of the present application, the rotation speed of the reduction reaction is 0-180rpm, preferably 120-180rpm.

[0060] According to the present application, the biosynthesis method further comprises a step of separating and purifying the gold nanoparticles and / or gold nanoparticle-biocatalyst hybrid, and the gold nanoparticles and / or gold nanoparticle-biocatalyst hybrid in the bacterial solution containing gold nanoparticles are separated and purified to obtain gold nanoparticle products and / or gold nanoparticle-biocatalyst hybrid products.

[0061] In the present application, there is no particular limitation or requirement for the separation and purification of free gold nanoparticles, as long as free gold nanoparticle monomers can be obtained. For example, since the gold nanoparticles and gold nanoparticle-biocatalyst hybrid will self-precipitate in the bacterial solution containing gold nanoparticles, the gold nanoparticles and gold nanoparticle-biocatalyst hybrid can be separated and purified by centrifugal separation, and the carbon component (i.e. biocatalyst) in the gold nanoparticle-biocatalyst hybrid can be burned off by burning to obtain free gold nanoparticle products.

[0062] The gold nanoparticle-biocatalyst hybrid in the present application is not particularly limited or required in separation and purification, as long as the gold nanoparticle-biocatalyst hybrid can be separated and obtained. For example, the gold nanoparticles and the gold nanoparticle-biocatalyst hybrid can be separated and purified by centrifugal separation, because the gold nanoparticles and the gold nanoparticle-biocatalyst hybrid can be self-precipitated in the bacterial solution containing the gold nanoparticles.

[0063] The skilled person in the art should understand that the gold nanoparticle-biocatalyst hybrid in the present application can be directly inoculated into the fermentation system for hydrogen production without separation when the gold nanoparticle-biocatalyst hybrid is used for hydrogen production.

[0064] The gold nanoparticles prepared by the biosynthesis method of the first aspect of the present application in the second aspect of the present application are mainly free gold nanoparticles prepared by the above-mentioned biosynthesis method of gold nanoparticles.

[0065] The research results show that the shape and size of the gold nanoparticles prepared by the method of the present application can be regulated by the reaction time, and the gold nanoparticles can be directly applied to the biological catalytic reaction of the gold nanoparticle synthesis bacteria (biological catalyst for synthesizing gold nanoparticles), thereby significantly improving the production capacity of the target product of the gold nanoparticle synthesis bacteria (biological catalyst for synthesizing gold nanoparticles). For example, when the strict anaerobic hydrogen-producing bacterium Clostridium butyricum is used as the biological catalyst for biosynthesis of gold nanoparticles, the gold nanoparticles with uniform particle size and regular shape (0.5 mM chloroauric acid in a physiological saline system) can be synthesized, and the gold nanoparticles synthesized by the Clostridium butyricum can significantly promote the anaerobic hydrogen production capacity of the Clostridium butyricum under light. The gold nanoparticle synthesis method of the present application has the advantages of simple operation and environmental friendliness, and the synthesized gold nanoparticles have the advantages of high biocompatibility and catalytic activity.

[0066] The free gold nanoparticles provided in the present application can be applied to biological hydrogen production and other uses.

[0067] The gold nanoparticle-biocatalyst hybrid prepared by the biosynthesis method of the first aspect of the present application in the third aspect of the present application is composed of gold nanoparticles uniformly attached and distributed on the entire biocatalyst.

[0068] Preferably, the biocatalyst comprises one or more of Clostridium butyricum, Clostridium acetobutylicum, Clostridium beijerinckii, Enterobacter aerogenes and Escherichia coli, and more preferably Clostridium butyricum.

[0069] The application of the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application in the biological hydrogen production can be understood as a method for the biological hydrogen production using the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application, and can also be understood as a method for the hydrogen production by the enhanced dark / light fermentation using the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application.

[0070] According to the present application, the application comprises the step of preparing a hydrogen production culture system containing the gold nanoparticles, which comprises mixing the gold nanoparticles and the hydrogen production bacteria into a hydrogen production culture solution to form the hydrogen production culture system containing the gold nanoparticles.

[0071] Preferably, the hydrogen production bacteria are Clostridium butyricum, and correspondingly, the hydrogen production culture solution is a Clostridium butyricum hydrogen production culture solution. Therefore, the above-mentioned application can also be understood as the application of the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application in the hydrogen production by Clostridium butyricum.

[0072] In some preferred embodiments of the present application, the step of preparing the hydrogen production culture system containing the gold nanoparticles comprises mixing the bacteria solution containing the gold nanoparticles prepared by the biosynthesis method according to the first aspect of the present application into a hydrogen production culture solution to form the hydrogen production culture system containing the gold nanoparticles; wherein the gold nanoparticles exist in the bacteria solution containing the gold nanoparticles in the form of gold nanoparticle-Clostridium butyricum hybrid.

[0073] Preferably, the inoculation amount of the bacteria solution containing the gold nanoparticle-Clostridium butyricum hybrid in the hydrogen production culture system is 5%-20% of the total volume of the fermentation system, preferably 10%-20%.

[0074] According to the present application, the application further comprises the step of biological hydrogen production culture, which comprises carrying out the biological hydrogen production culture on the hydrogen production culture system containing the gold nanoparticles to produce hydrogen.

[0075] Preferably, the light source for the biological hydrogen production culture comprises one or more of LED visible light, natural light and xenon lamp.

[0076] In some embodiments of the present application, the illumination intensity for the biological hydrogen production culture is 0-5mW / cm 2 , preferably 2.5mW / cm 2 .

[0077] In some embodiments of the present application, the biosynthesis method of the gold nanoparticles comprises: using chloroauric acid as a gold nanoparticle precursor, using Clostridium butyricum as a biological catalyst, and oscillating and culturing the chloroauric acid and Clostridium butyricum in a Clostridium butyricum culture solution for a certain period of time, so that a bacterial solution containing a gold nanoparticle-Clostridium butyricum hybrid and a small amount of free gold nanoparticles can be obtained. After centrifugation (5000 rpm for 5 min), the hybrid system is inoculated in a Clostridium butyricum hydrogen production culture medium for photofermentation, and the hydrogen production capacity of the hybrid system is significantly higher than that of bare Clostridium butyricum. The gold nanoparticle preparation method has the advantages of simple operation, green environmental protection, and high biocompatibility. This fully shows that the gold nanoparticles prepared by the present application can be applied to enhance the photofermentation hydrogen production of hydrogen-producing bacteria.

[0078] The detection and calculation method involved in the present application is as follows:

[0079] Gold ion reduction efficiency = (total gold concentration - free gold concentration) * 100 / total gold concentration

[0080] In the present application, the total gold concentration in the culture solution is determined by 725ES type Agilent ICP-OES. When measuring the free gold concentration, the bacterial solution containing the gold nanoparticle-Clostridium butyricum hybrid is first centrifuged at 5000 rpm for 5 min, and the supernatant after centrifugation is used to determine the free gold element concentration in the bacterial solution by 725ES type Agilent ICP-OES.

[0081] The bacterial culture formula involved in the present application is as follows:

[0082] (1) LB medium formula (1L) for E. coli and Enterobacter aerogenes: tryptone 10 g / L, yeast extract 5 g / L; sodium chloride 10 g / L, add 1L deionized water, autoclave 121℃ for 20 min or 116℃ for 25 min, and naturally cool to room temperature for use as an enterobacterium culture solution.

[0083] (2) Strengthened Clostridium medium formula (1L): yeast powder 3g, beef powder 10g, peptone 10g, soluble starch 1g, glucose 5g, sodium acetate 3g, NaCl 5g, L-cysteine hydrochloride 0.5g, add 1L deionized water, pH=6.8±0.2, autoclave 121℃ for 20 min or 116℃ for 25 min, and naturally cool to room temperature for use as a Clostridium culture solution.

[0084] (3) Improved Clostridium sp. culture medium formula (1L): yeast powder 3g, beef powder 10g, proteose peptone 10g, soluble starch 1g, glucose 10g, sodium acetate 3g, NaCl 5g, L-cysteine hydrochloride 1.5g, add 1L deionized water, pH = 6.8 ± 0.2, autoclave sterilization 121℃ for 20min or 116℃ for 25min, natural cooling to room temperature as the culture solution for hydrogen production by fermentation.

[0085] III. Examples

[0086] The present application is specifically illustrated by specific examples as follows. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, can be obtained from commercial channels.

[0087] The following examples relate to the following strains:

[0088] Escherichia coli JM109 (BNCC353808), North China Bioengineering- Henan Industrial Microbial Strain Engineering Research Center.

[0089] Enterobacter aerogenes (CGMCC: 1.4539), China General Microbiological Culture Collection Center.

[0090] Clostridium acetobutylicum (CGMCC: 1.5074), China General Microbiological Culture Collection Center.

[0091] Clostridium beijerinckii (CGMCC: 1.5077), China General Microbiological Culture Collection Center

[0092] Clostridium butyricum (CICC: 23847), China Industrial Microbial Strain Preservation and Management Center.

[0093] Example 1: Morphology of gold nanoparticles synthesized by reduction of chloroauric acid by Clostridium butyricum

[0094] After centrifuging 20 mL of Clostridium butyricum culture in liquid enhanced Clostridium medium for 12 h at 5000 rpm for 5 min, discard the supernatant, add 20 mL of sterile enhanced Clostridium medium or sterile physiological saline, and add filtered sterile chloroauric acid solution to make the concentration of chloroauric acid 0.5 mM or 2 mM and the OD600 of the bacterial culture 2.74. After sealing, incubate statically in a 37 ℃ incubator for 24 h. After centrifuging the bacterial suspension containing gold nanoparticles at 5000 rpm for 5 min, it was added to a cooled 2.5% glutaraldehyde fixative and refrigerated at 4℃ for 12 h. Then, the 2.5% glutaraldehyde fixative was discarded, and the sample was washed three times with 100 mM, pH 7.4 PBS buffer for 15 min each time. The sample was then dehydrated with ethanol-water solutions of different concentrations (30%, 50%, 70%, 80%, 90% and anhydrous ethanol) for 15 min each time. Finally, it was treated with 100% ethanol for 20 min. The bacterial suspension was then dropped onto a copper grid, dried, and its morphology was observed in a JEOL JEM-F200 transmission electron microscope.

[0095] The results are as follows Figure 1 As shown, in the chloroauric acid-free system, Clostridium butyricum has a smooth surface and no black particles are present [see...]. Figure 1 (a)]. After culturing a mixture of Clostridium butyricum containing 0.5 mM chloroauric acid for 24 h, obvious black gold nanoparticles were observed on the surface of Clostridium butyricum under bright-field transmission electron microscopy [see...]. Figure 1 (b)], and the entire Clostridium butyricum cell exhibited the characteristic brilliance of noble metal elements in the dark field of transmission electron microscopy, indicating that the gold nanoparticles synthesized by Clostridium butyricum were uniformly distributed throughout the Clostridium cell [see Figure 1 (c)], and the size of the gold nanoparticles is so small that no obvious black gold particles can be observed. Figure 1 (d) shows the morphology of gold nanoparticles generated by the reduction of Clostridium butyricum after increasing the concentration of chloroauric acid by 4 times (2 mM). Obvious black spherical gold particles with a diameter of approximately 100 nm can be observed, and obvious pores are present on the surface of Clostridium butyricum, indicating that the spherical gold nanoparticles have detached from the cells. After culturing Clostridium butyricum mixed with 0.5 mM chloroauric acid in physiological saline for 24 h, obvious spherical black gold nanoparticles were observed on the surface of Clostridium butyricum under bright-field transmission electron microscopy [see...]. Figure 1 (e)], with a particle size of about 20 nm, and gold nanoparticles exhibiting characteristic bright light in the dark field of transmission electron microscopy, are distributed throughout the cell surface, indicating that the gold nanoparticles synthesized by Clostridium butyricum in physiological saline are uniformly distributed throughout the Clostridium cell surface [see Figure 1 (f)]. Figure 1(g) is the morphology of the gold nanoparticles reduced by C. butyricum in normal saline after the concentration of chloroauric acid is increased by 4 times (2 mM). There are no holes on the cell surface, and part of the synthesized gold nanoparticles is still attached to the cell surface. However, the shape of the gold nanoparticles is diversified, including not only spherical shape but also triangular, pentagonal and hexagonal shapes. Figure 1 It is shown that the C. butyricum cells in the fortified Clostridium medium and normal saline can both reduce chloroauric acid to generate gold nanoparticles. When the concentration of chloroauric acid is 0.5 mM, the gold nanoparticles synthesized in the fortified Clostridium medium have very small particle size, while the gold nanoparticles in normal saline have uniform particle size, are mostly regular spherical shape, and are mostly attached to the Clostridium cells. When the concentration of chloroauric acid reaches 2 mM, the gold nanoparticles reduced in the fortified Clostridium medium have fallen off the cell surface and are regular spherical nanoparticles. However, the gold nanoparticles reduced in normal saline are still partially attached to the cell surface, and the shape of the gold nanoparticles is diversified.

[0096] Example 2: Reduction effect of different microorganisms on gold ions

[0097] After 20 mL of E. coli, Enterobacter aerogenes, C. acetobutylicum, C. butyricum and C. beijerinckii cultured in the culture medium (LB medium for E. coli and Enterobacter aerogenes, and fortified Clostridium medium for C. acetobutylicum, C. butyricum and C. beijerinckii) for 12 h were centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and fresh sterile liquid medium (LB medium for E. coli and Enterobacter aerogenes, and fortified Clostridium medium for C. acetobutylicum, C. butyricum and C. beijerinckii) and filtered chloroauric acid were added to make the concentration of chloroauric acid 0.5 mM. Then, the system was placed in a 37°C incubator for static culture, and the system of C. acetobutylicum, C. butyricum and C. beijerinckii was strictly sealed. After 24 h of culture, the bacterial liquid containing self-precipitated gold nanoparticles was taken, and the bacterial liquid and the supernatant (obtained by centrifugation at 5000 rpm for 5 min) containing gold nanoparticles were measured for total gold element concentration and free gold element concentration by Agilent 725ES ICP-OES, respectively, and the reduction efficiency of different microorganisms on gold ions was further calculated.

[0098] The results are shown in Table 1. Figure 2 As shown in Table 1, different microorganisms have good reduction ability on gold ions. After 1 mM of chloroauric acid was co-cultured with the five microorganisms for 24 h, the reduction efficiency of E. coli, Enterobacter aerogenes and C. acetobutylicum on gold ions was all above 96%, and the reduction efficiency of C. butyricum and C. beijerinckii on gold ions was about 88%.

[0099] Example 3: Reduction effect of C. butyricum on different concentrations of gold ions

[0100] The 20 mL of Clostridium butyricum liquid culture in liquid reinforced Clostridium medium for 12 h was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. 20 mL of sterile fresh liquid reinforced Clostridium medium and a sterile filtered chloroauric acid solution were added to make the concentration of chloroauric acid 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM, respectively. After sealing and incubation at 37°C in an incubator for 24 h, the bacterial liquid containing the gold nanoparticle-biocatalyst hybrid was obtained. The bacterial liquid containing gold nanoparticles and the supernatant (the bacterial liquid containing gold nanoparticles was centrifuged at 5000 rpm for 5 min to obtain the supernatant) were measured for total gold element concentration and free gold element concentration, respectively, by Agilent 725ES ICP-OES, and the reduction efficiency of Clostridium butyricum to gold ions was further calculated.

[0101] The results are shown in Figure 3 As the concentration of chloroauric acid increased, the reduction efficiency of Clostridium butyricum to gold ions first increased and then decreased, and when the concentration of gold ions was 0.5 mM, the reduction efficiency of Clostridium butyricum to gold ions was the highest, about 88.36% of gold ions were reduced, and when the concentration of gold ions increased to 2 mM, the reduction efficiency of Clostridium butyricum to gold ions decreased to 32.43%. Therefore, in the system of Clostridium butyricum reducing gold ions to generate gold nanoparticles, the optimal working concentration of gold ions was 0.5 mM.

[0102] Example 4: Reaction time of Clostridium butyricum reducing gold ions

[0103] The 20 mL of Clostridium butyricum liquid culture in liquid reinforced Clostridium medium for 12 h was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. 20 mL of sterile fresh liquid reinforced Clostridium medium or 20 mL of sterile physiological saline and a sterile filtered chloroauric acid solution were added to make the concentration of chloroauric acid 0.5 mM. After sealing and incubation at 37°C in an incubator, the bacterial liquid containing self-precipitated gold nanoparticles was taken at regular intervals (6 h, 12 h, 24 h, 36 h, and 48 h). The bacterial liquid containing gold nanoparticles and the supernatant (centrifuged at 5000 rpm for 5 min to obtain the supernatant) were measured for total gold element concentration and free gold element concentration, respectively, by Agilent 725ES ICP-OES, and the reduction efficiency of Clostridium butyricum to gold ions was further calculated.

[0104] The results are shown in Figure 4As shown, the reduction efficiency of Clostridium butyricum to gold ions in both physiological saline and reinforced Clostridium medium increased first and then remained unchanged with the extension of reduction time. After the reaction reached equilibrium, the reduction efficiency of Clostridium butyricum to gold ions in physiological saline was slightly higher than that in the reinforced Clostridium medium system. The reduction efficiency of Clostridium butyricum to gold ions in the reinforced Clostridium medium system was 88% after 24 h of reduction, while the reduction efficiency of Clostridium butyricum to gold ions in physiological saline increased to 96.06% under the same reduction time. The reduction efficiency of Clostridium butyricum to gold ions did not increase significantly when the reaction time was further extended to 48 h. Therefore, the reduction time of Clostridium butyricum to gold ions was 6-48 h, and the preferred reduction time was 24 h.

[0105] Example 5: Hydrogen production by anaerobic photobiological fermentation of the mixture of gold nanoparticles and Clostridium butyricum.

[0106] In a strict anaerobic photobiological fermentation system of 100 mL liquid reinforced Clostridium medium, glucose (working concentration of 10 g / L) and cysteine (working concentration of 1.5 g / L) were supplemented as carbon source and exogenous electron donor, respectively. The fermentation system was inoculated with the hybrid containing Clostridium butyricum reduced gold nanoparticles prepared in Example 1 in the reinforced Clostridium medium with 0.5 mM chloroauric acid as the precursor (the supernatant was discarded by centrifugation, and fresh liquid reinforced Clostridium medium was added to the original volume), and the inoculation amount was 10% (volume fraction, OD600=0.220) of the fermentation system. The photobiological fermentation hydrogen production system was placed in a light incubator at 37°C, and the photobiological fermentation culture was carried out under LED white light (2.5 mW / cm 2 ) (120 rpm), and a dark fermentation system wrapped with tin paper was set as a control. After 2 h of culture, the hydrogen production system was connected to a gas bag, and the gas was collected at fixed time intervals. The total volume of biogas produced by fermentation was determined by the drainage method, and the concentration of hydrogen contained in the biogas was further determined by gas chromatography (TCD detector, Shimadzu) to calculate the total amount of hydrogen produced by fermentation.

[0107] As Figure 5As shown, the cumulative hydrogen production of Clostridium butyricum dark fermentation for 144h was 1289.49mL / L, and the cumulative hydrogen production of Clostridium butyricum photo-fermentation was increased to 1417.11mL / L. The hydrogen production of the dark fermentation and the photo-fermentation of the hybrid of the gold nanoparticles generated by Clostridium butyricum reducing gold ions and Clostridium butyricum were obviously higher than that of the naked Clostridium butyricum system. The cumulative hydrogen production of the photo-fermentation of the gold nanoparticles and Clostridium butyricum was increased by 66.5% compared with the dark fermentation of the naked Clostridium butyricum, reaching 2147.40mL / L. It was shown that the gold nanoparticles generated by Clostridium butyricum reducing gold ions could promote the photo-fermentation of Clostridium butyricum to produce hydrogen. Therefore, the gold nanoparticles generated by Clostridium butyricum reducing gold ions could be applied to enhance the dark fermentation or the photo-fermentation of Clostridium butyricum to produce hydrogen by the method of the present application. Further, the gold nanoparticles generated by the present application could be used to enhance the ability of the host cell (the cell for synthesizing gold nanoparticles) to produce target substances by photo-biological catalysis.

[0108] It should be noted that the above-described embodiments are merely preferred embodiments of the present application, which are used to explain the present application and do not constitute any limitation to the present application. The present application is described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified within the scope of the claims of the present application, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein, but rather, the present application can be extended to all other methods and applications having the same function.

Claims

1. Use of gold nanoparticle-biocatalyst hybrid in biohydrogen production, characterized in that, The gold nanoparticle-containing bacteria solution is mixed with hydrogen production culture solution to form a gold nanoparticle-containing hydrogen production culture system, and the system is subjected to biological hydrogen production culture to produce hydrogen; In the gold nanoparticle-containing bacteria solution, the gold nanoparticles exist in the form of gold nanoparticle-biological catalyst hybrid, and the hydrogen production culture system contains an exogenous electron donor cysteine with a working concentration of 1.5 g / L; The light source of the biological hydrogen production culture includes one or several of LED visible light, natural light and xenon lamp; the light intensity of the biological hydrogen production culture is 2.5mW / cm 2 ; The preparation method of the gold nanoparticle-biological catalyst hybrid is as follows: gold ions as gold nanoparticle precursors are mixed with biological catalysts and reduction reaction solvents to perform reduction reaction of the gold ions, and a bacteria solution containing gold nanoparticles is obtained, wherein the gold nanoparticles exist in the form of gold nanoparticle-biological catalyst hybrid, the gold ions are chloroauric acid, the working concentration of the gold ions is 0.5 mmol / L, the biological catalysts are Clostridium butyricum with a working concentration of OD600 value of 0.1-5, and the reduction reaction solvents are physiological saline or a reinforced Clostridium culture medium, the reduction reaction time is 24 h, and the reduction reaction temperature is 37-40°C; The formula of the reinforced Clostridium culture medium is as follows: 3 g of yeast powder, 10 g of beef powder, 10 g of peptone, 1 g of soluble starch, 5 g of glucose, 3 g of sodium acetate, 5 g of NaCl, 0.5 g of L-cysteine hydrochloride, 1 L of deionized water, and pH=6.8±0.

2.

2. Use according to claim 1, characterized in that, The inoculation amount of the gold nanoparticle-containing bacteria solution in the hydrogen production culture system is 10%-20% of the total volume of the fermentation system.

3. Use according to claim 1, characterized in that, The rotation speed of the reduction reaction is 120-180 rpm.

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