CN115261269A - Culture medium for xylose colatopsis and method for preparing bacterial cellulose by using culture medium - Google Patents
Culture medium for xylose colatopsis and method for preparing bacterial cellulose by using culture medium Download PDFInfo
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Abstract
The invention provides a xylose colatole type bacillus culture medium and a method for preparing bacterial cellulose by using the culture medium. The culture medium is obtained by adding PET ammonolysis solution based on a formula of an HS culture medium, the ammonolysis solution is used as an accelerant to effectively improve the cellulose yield of the Xylobacter colatole by 2 times, and the activity of related enzymes such as UDP-glucose pyrophosphorylase and glucose phosphoglucomutase is improved by regulating and controlling the activity of the cellulose synthesis, the activity reduction of related enzymes such as pyruvate kinase and phosphofructokinase in a glucose decomposition way is inhibited, so that the yield of the bacterial cellulose is improved to 4.91g/L. The bacterial cellulose prepared by the method has lower crystallinity, better surface area and larger pore diameter. The method for preparing the fermented PET carbon source by using the waste PET as the carbon source of the fermented bacterial cellulose after the waste PET is treated solves the problem of treatment of the waste PET, reduces the fermentation production cost, saves resources and reduces the environmental pollution.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a xylose colal type bacillus culture medium and a method for preparing bacterial cellulose by using the culture medium.
Background
Polyethylene terephthalate (PET) is a common synthetic fiber with desirable characteristics of fabrics and packaging materials, such as moldability, transparency, and durability. It is reported that 3.59 million tons of PET are prepared globally in 2020, which is expected to increase by 3% each year in the future. In recent years, PET has gained increasing attention due to its low biodegradability and photodegradation. Traditional approaches to waste management result in the release of large quantities of PET into the environment, which is a serious environmental problem. For example, scattered PET waste blocks drainage, deteriorates soil properties, and emits toxic gases upon combustion, thereby disturbing the environment. Furthermore, PET waste constitutes a serious health risk for plants, animals and humans. Different strategies have been developed to recover waste PET or convert it into other useful products. However, only less than 30% of PET is recycled due to high processing costs, high consumer acceptance, safety management, and the like.
Bacterial Cellulose (BC) is a white gelatinous BC particle secreted by some gram-negative bacteria, produced at the air-liquid interface, that is of great interest to material scientists and polymer chemists due to its unique properties, such as high purity (free of pectin, lignin and hemicellulose), crystallinity (> 80%). Young's modulus (about 25 GPa), tensile strength (200-400 MPa), thermal stability (thermal degradation temperature of 355-375 ℃) and ultrafine three-dimensional (3D) cellulose nanofiber networks (2-9 nm). BC is widely explored as an important biomaterial due to its unique properties, and is used in many fields such as food, cosmetics, biomedicine, textiles, and high-end acoustic membranes. Although BC manufacturers produce BC using various substrates, their conversion efficiency is low, resulting in low yield, resulting in high production cost, thereby limiting their wide application. In order to improve the substrate conversion efficiency of BC producers and minimize the production cost of BC, hydrolysates of agricultural wastes, such as wheat straw, sunflower seed meal and colored rice, are rich sources of different sugars, which have been converted into BC. During the de novo synthesis of BC, some by-products, such as organic acids based on alpha-glucuronic acid, are also produced, which can reduce the overall yield of BC.
How to use carbon-rich PET plastics as raw material for biotechnological processes instead of waste is a viable alternative to improve the recycling of PET. At present, no report that PET plastics are directly used as additives to promote bacterial cellulose synthesis exists.
Disclosure of Invention
The invention aims to provide a xylose colatole type bacillus culture medium and a method for preparing bacterial cellulose by using the culture medium, aiming at the defects of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention aims to provide a xylose colal type bacillus culture medium, wherein each liter of the culture medium contains 20g of glucose, 5g of yeast extract, 5g of peptone, 1.15g of citric acid monohydrate, 2.7g of disodium hydrogen phosphate and the balance of polyethylene glycol terephthalate ammonolysis solution, and the pH value is 6.5-7.0.
Further, the preparation process of the polyethylene glycol terephthalate ammonolysis solution comprises the steps of finishing the polyethylene glycol terephthalate into small blocks, and then adding 0.4-0.8 mol/L NH3·H2And (3) hydrolyzing in O, wherein the hydrolysis reaction temperature is 160-180 ℃, the reaction time is 25-30 min, and after the reaction is finished, the reaction solution is neutralized to the pH value of 6.5-7.5 to obtain the polyethylene terephthalate ammonolysis solution.
The second object of the present invention is to provide a method for preparing bacterial cellulose using the aforementioned xylose colal bacterium culture medium, comprising the steps of:
step S1, activation of Xylobacter colatole
Inoculating the strain of the xylose colt-shaped bacillus onto a solid culture medium to obtain a colony of the xylose colt-shaped bacillus;
step S2, the enlarged culture of the xylose foal bacillus
Inoculating the bacterial colony of the bacillus foeniculi obtained in the step S1 on a liquid culture medium to obtain a bacillus foeniculi seed solution;
step S3, preparation of bacterial cellulose fermentation medium
Adopting the xylose coltfoal-shaped bacillus culture medium;
step S4, preparation of bacterial cellulose fermentation liquor
Inoculating the xylose coltsfoot bacterium seed liquid obtained in the step S2 into the bacterial cellulose fermentation culture medium obtained in the step S3 according to the inoculation amount of 5-15%, and fermenting for 8-10 days at the temperature of 28-30 ℃ to obtain bacterial cellulose fermentation liquor;
step S5, preparation of bacterial cellulose
Taking out the bacterial cellulose membrane on the upper layer of the liquid surface of the bacterial cellulose fermentation liquid obtained in the step S4, firstly adding the bacterial cellulose membrane into a sodium carbonate solution, heating and boiling the bacterial cellulose membrane, and then washing the bacterial cellulose membrane with water; and then putting the bacterial cellulose membrane into water, heating and boiling, finally washing to obtain a pure bacterial cellulose membrane, washing to be neutral, sucking water, and drying to be constant weight to obtain the bacterial cellulose.
Further, in step S1, the solid medium contains 5 to 6g of yeast extract, 5g of peptone, 15 to 20g of glucose, 1 to 1.15g of citric acid monohydrate, and 2.5 to 2.7g of Na2HPO415-18.0 g of agar and 1.0L of distilled water.
Further, in step S1, the liquid culture medium contains 5-6 g/L yeast extract, 4-5 g/L peptone, 15-20 g/L glucose, 1-1.15 g/L citric acid monohydrate, 2.5-2.7 g/LNa2HPO4The pH value is 6.5-7.0.
Further, in step S5, the concentration of the sodium carbonate solution is 1-3%.
Further, in step S5, the heating temperature is 80 ℃ to 100 ℃.
The third purpose of the invention is to provide bacterial cellulose prepared by the method.
Furthermore, the specific surface area of the bacterial cellulose is 33 to 37m2(ii)/g, the average pore diameter is 19 to 29nm.
Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:
(1) The invention discloses a method for promoting bacterial cellulose synthesis by using PET ammonolysis liquid, wherein a culture medium for bacterial cellulose fermentation is obtained by adding polyethylene terephthalate ammonolysis liquid (PETAH) as an accelerant on the basis of a formula of an HS culture medium, the cellulose yield of Xylobacter colanensis (Taonella. Copensis) is effectively improved by 2 times by using the polyethylene terephthalate ammonolysis liquid as the accelerant, and the activity of enzymes related to cellulose synthesis is improved, for example: the activities of UDP-glucose pyrophosphorylase (UGP) and glucose Phosphoglucomutase (PGM) are respectively improved by 30.63 percent and 135.24 percent, and related enzymes of a glucose decomposition pathway such as: the Pyruvate Kinase (PK) and Phosphofructokinase (PFK) are reduced by 40.34 percent and 52.63 percent, thereby improving the yield of the bacterial cellulose to 4.91g/L.
(2) The Bacterial Cellulose (BC) prepared by the method has lower crystallinity, better surface area and larger pore diameter, and compared with the method for fermenting and culturing the Xylothrix colatopsis (T.metpensis) by adopting the HS culture medium, the specific surface area of the bacterial cellulose prepared by the method is improved by about 3.9 times, and the total pore volume is increased by about 4 times.
(3) According to the invention, the polyethylene glycol terephthalate is effectively decomposed by a high-temperature ammonolysis method, the obtained polyethylene glycol terephthalate ammonolysis solution is used as a nutritional additive for microbial fermentation and is added into the traditional HS culture medium to obtain the bacterial cellulose culture medium, the polyethylene glycol terephthalate is effectively used as the additive, the pollution of the polyethylene glycol terephthalate to the environment is reduced, the cost for preparing the bacterial cellulose is also saved, and the bacterial cellulose culture medium is suitable for large-scale production of the bacterial cellulose.
Drawings
FIG. 1A is a graph showing the trend of PETAH concentration on DCW and BC production in PETAH-HS medium;
FIG. 1B is a graph comparing PETAH concentration versus glucose consumption and BC/glucose conversion in PETAH-HS medium;
FIG. 2 is a comparative analysis chart of growth process of Xylella xylocolae in PETAH-HS medium and HS medium; (A) a pH value change trend graph of a PETAH-HS culture medium and a HS culture medium, (B) a cell density change trend graph of the xylose colt bacillus cultured by the PETAH-HS culture medium and the HS culture medium, (C) a glucose change trend graph of the xylose colt bacillus cultured by the PETAH-HS culture medium and the HS culture medium, (D) a glucose change trend graph of the xylose colt bacillus cultured by the PETAH-HS culture medium and the HS culture medium, (E) a trend graph of Terephthalic Acid (TA) and Ethylene Glycol (EG) of the xylose colt bacillus cultured by the PETAH-HS culture medium and the HS culture medium, and (F) a change trend graph of bacterial cellulose yield of the xylose colt bacillus cultured by the PETAH-HS culture medium and the HS culture medium;
FIG. 3A is a comparison of IR spectra of BC samples produced in different media;
FIG. 3B is a graph comparing X-ray diffraction profiles of BC samples produced with different media;
FIG. 3C is a TGA thermogram of BC samples produced with different media;
FIG. 3D is a DTG thermal analysis comparison of BC samples produced by different media;
FIG. 4 is a graph showing the effect of different media on the involvement of BC anabolism and glucose catabolism, wherein (A) a graph showing the effect of Phosphofructokinase (PFK) activity, (B) a graph showing the effect of Pyruvate Kinase (PK) activity, (C) a graph showing the effect of Phosphoglucomutase (PGM) activity, and (D) a graph showing the effect of UDP-glucose pyrophosphorylase (UGP) activity.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in further detail below with reference to specific examples and accompanying drawings. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.
The strains used in the invention are as follows: the biological collection number of the xylose colatole bacillus (T.mepenis) is as follows: CICC10529.
Example 1
Preparing bacterial cellulose:
1. activation of xylaria coll (t. Mepenis)
0.5mL of sterile water was injected into the lyophilization tube and gently pipetted to dissolve well into a suspension. Sucking the bacterial suspension, injecting 200 mu L of the bacterial suspension into the surface of a culture medium, culturing at 30 ℃, and growing new bacterial colonies on the solid culture medium of the xylose colt bacillus after two days. Solid culture medium of xylose colt bacillus: 5g of yeast extract, 5g of peptone, 20g of glucose, 1.15g of citric acid monohydrate, 2.7g of Na2HPO4Agar 18.0g, distilled water 1.0L.
2. Expanded culture of xylaria coll (T.mepenis)
The formula for preparing the liquid culture medium of the xylose colatole comprises 5g/L of yeast extract, 5g/L of peptone, 20g/L of glucose, 1.15g/L of citric acid monohydrate and 2.7g/LNa2HPO4Adjusting pH to 7.0, inoculating activated Xylobacter colatoides in the culture medium, and shake culturing at 30 deg.C for 24 hr to obtain Xylobacter colatoides (T.meensis) seed solution with concentration of 106Per ml to 108One per mL.
3. Preparation of bacterial cellulose fermentation medium
The preparation process of the polyethylene terephthalate ammonolysis solution (PETAH) comprises the following steps: pulverizing polyethylene terephthalate into 3 × 3mm2Small pieces, then 0.6mol/L NH is added3·H2And (3) hydrolyzing in O, wherein the hydrolysis reaction temperature is 180 ℃, the reaction time is 30min, and after the reaction is finished, the reaction solution is neutralized to pH 7.0 to obtain the polyethylene terephthalate ammonolysis solution (PETAH).
1 percent of the total weight is takenAdding 5g of yeast extract, 5g of peptone, 20g of glucose, 1.15g of citric acid monohydrate and 2.7g of Na into the polyethylene terephthalate ammonolysis solution2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times until the bacterial cellulose membrane is neutral to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 4.91g/L.
Example 2
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of xylaria coll (T.mepenis)
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The preparation process of the polyethylene terephthalate ammonolysis solution (PETAH) comprises the following steps: the polyethylene terephthalate is arranged into 3 multiplied by 3mm2Small pieces, then add 0.4mol/L NH3·H2And (3) hydrolyzing in O, wherein the hydrolysis reaction temperature is 160 ℃, the reaction time is 25min, and after the reaction is finished, the reaction solution is neutralized to pH 7 to obtain polyethylene glycol terephthalate ammonolysis solution (PETAH).
1L 1.0% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparing bacterial cellulose:
taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC) per total volume of medium and was 3.07g/L.
Example 3
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of xylaria coll (T.mepenis)
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The preparation process of the polyethylene terephthalate ammonolysis solution (PETAH) comprises the following steps: the polyethylene terephthalate is arranged into 3 x 3mm2Small pieces, then 0.8mol/L NH was added3·H2And (3) hydrolyzing in O, wherein the hydrolysis reaction temperature is 170 ℃, the reaction time is 30min, and after the reaction is finished, the reaction solution is neutralized to pH 7 to obtain polyethylene glycol terephthalate ammonolysis solution (PETAH).
1L 1.0% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 4.58g/L.
Example 4
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of xylaria coll (T.mepenis)
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The polyethylene terephthalate ammonolysis solution was the same as in example 1.
1L 1.0% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 6.5 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the bacterial coltsfoot bacterium (T.mepenis) seed liquid, inoculating 15mL of the inoculum size per 85mL of the bacterial coltsfoot bacterium (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and after static fermentation is carried out for 8 days at 30 ℃, ending the fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 4.84g/L.
Example 5
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of T.mepensis
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The polyethylene terephthalate ammonolysis solution was the same as in example 1.
1L 1.0% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the bacterial coltsfoot bacterium (T.mepenis) seed liquid, inoculating 5mL of the inoculum size per 95mL of the bacterial coltsfoot bacterium (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and performing static fermentation at 28 ℃ for 8 days to finish the fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 3.58g/L.
Example 6
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of T.mepensis
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The polyethylene terephthalate aminolysis solution was the same as in example 1.
1L of 0.5% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 3.61g/L.
Example 7
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of xylaria coll (T.mepenis)
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The polyethylene terephthalate aminolysis solution was the same as in example 1.
Adding yeast extract 5g into 1L 1.5% polyethylene glycol terephthalate ammonolysis solution5g of peptone, 20g of glucose, 1.15g of citric acid monohydrate and 2.7g of Na2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparation of bacterial cellulose fermentation liquor
Taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into a 200ml 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing with deionized water for several times, putting the bacterial cellulose membrane into 200ml deionized water, carrying out water bath at 100 ℃ for 30min, and washing with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as dry weight of Bacterial Cellulose (BC)/total volume of medium and was 3.89g/L.
Comparative example 1
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of T.mepensis
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
Adding 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na into 1L distilled water2HPO4And adjusting the pH value to 7.0 to obtain the bacterial fiber fermentation medium.
4. Preparing bacterial cellulose fermentation liquor:
taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in the bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into 200ml of 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing the bacterial cellulose membrane with deionized water for several times, putting the bacterial cellulose membrane into 200ml of deionized water, carrying out water bath at 100 ℃ for 30min, and washing the bacterial cellulose membrane with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was calculated as Bacterial Cellulose (BC) dry weight/total volume of medium and was 2.28g/L.
Comparative example 2
1. Activation of xylaria coll (t. Mepenis)
The same as in example 1.
2. Expanded culture of xylaria coll (T.mepenis)
The same as in example 1.
3. Preparation of bacterial cellulose fermentation medium
The polyethylene terephthalate ammonolysis solution was the same as in example 1.
1L of 2.0% polyethylene terephthalate ammonolysis solution was added with 5g yeast extract, 5g peptone, 20g glucose, 1.15g citric acid monohydrate and 2.7g Na2HPO4And adjusting the pH value to 6.8 to obtain the bacterial fiber fermentation medium.
4. Preparing bacterial cellulose fermentation liquor:
taking the xylose coltsia coltflorus (T.mepenis) seed liquid, inoculating 10mL of inoculum size per 90mL of the xylose coltsia coltflorus (T.mepenis) seed liquid into a bacterial cellulose fermentation culture medium, and statically fermenting at 28 ℃ for 10 days to finish fermentation to obtain the bacterial cellulose fermentation liquid.
5. Preparation of bacterial cellulose
Taking a bacterial cellulose membrane in bacterial cellulose fermentation liquor, putting the bacterial cellulose membrane into a 200ml 2% sodium carbonate solution, carrying out water bath at 100 ℃ for 30min, washing with deionized water for several times, putting the bacterial cellulose membrane into 200ml deionized water, carrying out water bath at 100 ℃ for 30min, and washing with deionized water for several times to obtain a pure bacterial cellulose membrane. The yield was 2.08g/L, calculated as dry weight of Bacterial Cellulose (BC) per total volume of medium.
To better illustrate that polyethylene terephthalate (PETAH) hydrolysate has a promoting effect on the yield of bacterial cellulose in a certain concentration range, the applicant conducted a comparative study on the yield of Bacterial Cellulose (BC) and the Dry Cell Weight (DCW) in a culture prepared in examples 1, 6, 7, 1 and 2, wherein the bacterial cellulose fermentation medium prepared in example 1 is abbreviated as PETAH-HS medium, and the bacterial cellulose fermentation medium prepared in comparative example 1 is abbreviated as HS medium.
Results as shown in fig. 1A, the Dry Cell Weight (DCW) and BC yield in culture increased with increasing PETAH concentration to 1%, followed by a dramatic decrease in yield. The results further show that when the original PETAH was diluted twice with water, the BC yield was up to 4.91g/L in PETAH-HS medium. In contrast, 2.28g/L BC was produced in HS medium, indicating a much higher yield in PETAH-HS medium. In the present invention, PETAH at optimal concentration increased BC production by 115%. The residual glucose content in PETAH-HS medium was also determined to calculate the efficiency of glucose conversion to BC during BC production.
As shown in fig. 1B, the percent of glucose consumption decreased from 96.05% to 85.03% as the PETAH content increased from 0 to 1%. As the yield of BC increases, the decrease in glucose consumption results in a significant increase in the efficiency of conversion of BC to glucose from 12.04% to 29.22%. This suggests that PETAH has been used to partially replace glucose as an energy source; therefore, more glucose was used for BC synthesis. However, as the PETAH concentration was further increased to 2%, the conversion efficiency of BC to glucose dropped dramatically to 5.47%, probably due to the growth inhibitory effect of high substrate concentration on t.mepensis, ultimately resulting in reduced BC yield.
To better illustrate the reason why the yield of the bacterial cellulose prepared by fermenting xylose colatobacter colatoides with the bacterial cellulose fermentation medium of the present invention is improved, the applicant studied the time course of bacterial cellulose secretion by xylose colatobacter colatorius (t.mepenis) with the bacterial cellulose fermentation medium (PETAH-HS medium) prepared in example 1, and compared the structural characteristics of bacterial cellulose produced in different media (PETAH-HS medium or HS medium).
The results are shown in FIG. 2, comparing the growth curves of cultures grown in HS medium and PETAH-HS medium, during the whole fermentation: as shown in figure (a), the pH of the xylose foal bacillus culture supplemented with PETAH was much higher than that of HS medium; as shown in panel (B), cell density was much higher in PETAH supplemented cultures; as shown in figures (C) and (D), when grown in 1% petah medium, the bacterium jujulian absorbs less glucose and produces less gluconic acid than the strains grown in HS medium; as shown in FIG. E, under the optimum culture conditions (1% in PETAH medium), the following were detected by liquid chromatography: the bacterium foal absorbs almost all of the Terephthalic Acid (TA) within 6 days, while Ethylene Glycol (EG) remains until day 8; as shown in FIG. F, the maximum BC yield of 4.91g/L obtained from PETAH-HS medium was much higher than the BC yield (2.28 g/L) obtained from HS medium.
As shown in FIG. 3A, a comparison of the IR spectra of BC samples produced for different media. The spectra from PETAH supplemented HS media are nearly similar to those obtained from BC-HS, showing characteristic peaks of cellulose at 556, 1054, 2895, and 3336cm-1, with no significant difference in FTIR spectra for the two samples. Both BC films showed peaks at 710 and 750cm-1, corresponding to the monoclinic I α and triclinic I β moieties present in type I cellulose. From the FTIR spectra, the I.alpha.fraction can be calculated using the Machado method, and low I.alpha.content is found in BC samples produced in PETAH-HS medium (BC-PETAH) compared to HS medium (BC-HS). This may be because the extender disturbs the formation of micro-fibers and crystallite orientation.
As shown in fig. 3B, X-ray diffraction profiles of BC samples produced for different media were compared. Both BC films prepared from two different media showed similar patterns with three diffraction peaks at 2 θ =14.5 °, 16.7 ° and 22.5 °, corresponding to (100), (010) and (110) lattice planes, respectively. This is all characteristic bands for the BC sample with classical cellulose (type I). The CI for BC-PETAH (73.17%) was lower than BC-HS (84.63%), probably due to the fact that the crystallization of BC could be disrupted by the addition of PETAH additive during the cultivation process. In addition, the additive PETAH changes the cross-linking hydrogen bonding network of BC, increases the distance between cellulose chains, and breaks the process of arranging linear beta-glucan chains into an ordered crystal structure.
As shown in FIG. 3C, a TGA thermogram of BC samples generated for different media is shown. TGA thermograms of different BC films show that thermal depolymerization of BC-PETAH (red) starts at 240 ℃ and thermal depolymerization of BC-HS (black) starts at 250 ℃.
As shown in fig. 3D, DTG thermograms of BC samples produced for different media. The maximum degradation temperatures of BC-PETAH and BC-HS were shown to be 298 ℃ and 320 ℃, respectively, indicating that BC-PETAH is less thermally stable than BC-HS. The maximal degradation of BC that occurs at this temperature may result in the release of levoglucosan.
The average polymerization Degree (DP) values of BC-HS and BC-PETAH are shown in Table 1. The DP of BC decreased after PETAH was added to HS medium. The DP of BC-PETAH was 1878, lower than BC-HS (2088), consistent with CI as determined by XRD.
The BC has well-known mechanical properties that make it suitable for use in a variety of applications, such as biomedicine, food additives and sewage purification. The effect of the additives on the mechanical behavior of BC was investigated by measuring young's modulus, tensile strength and elongation at break, and the data is shown in table 1. The applicant found that elongation at break of BC-PETAH is slightly longer than that of BC-HS (p > 0.05), while Young's modulus and tensile strength of BC-PETAH are slightly lower than that of BC-HS (p > 0.05). Small reductions in mechanical properties may be associated with reductions in DP and CI.
Specific surface area and pore size are key parameters for controlling the adsorption behavior of BC. The applicant performed BJH pore size and BET surface area analyses. The increase in pore surface size results in an increase in specific surface area, as evidenced by the pore size of BCPETAH and BC-HS in the present invention. Table 1 shows that the average pore diameters of BC-PETAH BC-HS are 24.526nm and 17.645nm, respectively. At the same time, the total pore volume of BC-HS and BC-PETAH increased from 0.049 to 0.211cm3(ii) in terms of/g. This results in a specific surface area of from 9.069m2Expansion of/g (BC-HS) to 35.388m2(BC-PETAH). The specific surface area of BC-PETAH is 3 of BC-HS9 times.
TABLE 1 comparison of the characteristics of BC produced by different fermentation broths
The applicant has also carried out studies of key enzymes of sugar metabolism in order to explore the molecular mechanism of PETAH in promoting cellulose biosynthesis. The results found that glucose catabolism-related enzymes: the activity of Pyruvate Kinase (PK) and Phosphofructokinase (PFK) is reduced, and sugar decomposition is inhibited. Meanwhile, cellulose synthesis key enzyme: the activity of UDP-glucose pyrophosphorylase (UGP) and glucose Phosphoglucomutase (PGM) is enhanced, and the synthesis of bacterial cellulose is accelerated.
As shown in FIG. 4, the effect of different media on the activities of PFK (A), PK (B), PGM (C) and UGP (D) involved in BC anabolism and glucose catabolism is shown. The HS medium added with PETAH respectively improves the activity of UDP-glucose pyrophosphorylase (UGP) and glucose Phosphoglucomutase (PGM) by 30.63 percent and 135.24 percent, and reduces the activity of Pyruvate Kinase (PK) and Phosphofructokinase (PFK) by 40.34 percent and 52.63 percent.
In summary, the addition of PETAH to conventional HS media is a very simple and effective method to increase BC production without significantly changing its characteristics. The addition of 1% of PETAH with the optimal dose to the HS medium improves the glucose conversion efficiency of the Bacillus focolatole (T.mepenis) to 29.22%, and further promotes the BC yield to be increased to 4.91g/L, which is equivalent to 2.15 times. The significant increase in BC production is due to the modulation of the activity of the four key enzymes (PFK, PK, PGM and UGP). The method provided by the present invention can improve BC yield using cost-effective PETAH as a nutritional additive.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
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, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. The culture medium for the xylose colt-shaped bacilli is characterized by comprising 20g of glucose, 5g of yeast extract, 5g of peptone, 1.15g of citric acid monohydrate, 2.7g of disodium hydrogen phosphate and the balance of polyethylene glycol terephthalate ammonolysis solution, wherein the pH value is 6.5-7.0.
2. The culture medium for xylofoal bacteria according to claim 1, wherein the ammonolyzed polyethylene terephthalate solution is prepared by cutting polyethylene terephthalate into small pieces and adding 0.4-0.8 mol/L NH3·H2And (3) hydrolyzing in O, wherein the hydrolysis reaction temperature is 160-180 ℃, the reaction time is 25-30 min, and after the reaction is finished, the reaction solution is neutralized to pH 6.5-7.5 to obtain the polyethylene glycol terephthalate ammonolysis solution.
3. A process for the preparation of bacterial cellulose using the culture medium of clostridium colatole as defined in claim 1 or 2, comprising the steps of:
s1, activation of Xylobacter colatole
Inoculating the strain of the bacillus colatole onto a solid culture medium to obtain a bacillus colatole colony;
s2, amplification culture of Xylobacter colatoides
Inoculating the bacterial colony of the xylose colatorium obtained in the step S1 on a liquid culture medium to obtain a seed solution of the xylose colatorium;
s3, preparation of bacterial cellulose fermentation medium
Using a culture medium of Xylomyces colatole according to claim 1 or 2;
s4, preparation of bacterial cellulose fermentation liquor
Inoculating the bacterial cellulose fermentation medium obtained in the step S3 with the bacterial coltfoal seed solution obtained in the step S2 according to the inoculation amount of 5% -15%, and fermenting for 8-10 days at 28-30 ℃ to obtain bacterial cellulose fermentation liquor;
s5, preparation of bacterial cellulose
Taking out the bacterial cellulose membrane on the upper layer of the liquid surface of the bacterial cellulose fermentation liquid obtained in the step S4, firstly adding the bacterial cellulose membrane into a sodium carbonate solution, heating and boiling the bacterial cellulose membrane, and then washing the bacterial cellulose membrane with water; and then putting the bacterial cellulose membrane into water, heating and boiling, finally washing to obtain a pure bacterial cellulose membrane, washing to neutrality, sucking water, and drying to constant weight to obtain the bacterial cellulose.
4. The method according to claim 3, wherein the solid medium comprises 5 to 6g of yeast extract, 5g of peptone, 15 to 20g of glucose, 1 to 1.15g of citric acid monohydrate, and 2.5 to 2.7g of Na in step S12HPO415-18.0 g of agar and 1.0L of distilled water.
5. The method according to claim 3, wherein in step S1, the liquid medium formulation contains 5-6 g/L yeast extract, 4-5 g/L peptone, 15-20 g/L glucose, 1-1.15 g/L citric acid monohydrate, 2.5-2.7 g/LNa2HPO4The pH value is 6.5-7.0.
6. The method of claim 3, wherein the concentration of the sodium carbonate solution in step S5 is 1-3%.
7. The method for preparing bacterial cellulose according to claim 6, wherein the heating temperature in step S5 is 80 ℃ to 100 ℃.
8. Bacterial cellulose obtainable by the method for the preparation of bacterial cellulose according to any one of claims 3 to 7.
9. The bacterial cellulose of claim 8, having a specific surface area of 33 to 37m2(iv) g, the average pore diameter is 19 to 29nm.
Priority Applications (1)
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| CN110343272A (en) * | 2019-07-09 | 2019-10-18 | 陕西师范大学 | A kind of bacteria cellulose nanofiber enhancing konjac glucomannan edible film and preparation method thereof |
| CN114127279A (en) * | 2019-07-12 | 2022-03-01 | 赢创运营有限公司 | Method for loading microorganisms on multiphase biological material |
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| CN114127279A (en) * | 2019-07-12 | 2022-03-01 | 赢创运营有限公司 | Method for loading microorganisms on multiphase biological material |
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