Gluconacetobacter xylinum for producing bacterial cellulose by efficiently fermenting lignocellulose hydrolysate and application of gluconacetobacter xylinum
By using Komagataeibacter xylinus, which is resistant to lignocellulose hydrolysate inhibitors, the problems of low productivity and high cost of bacterial cellulose are solved, efficient and low-cost bacterial cellulose production are achieved, and green production is promoted.
Patent Information
- Application Number
- CN202510032671.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-05-06
AI Technical Summary
The low productivity and high production cost of bacterial cellulose in the prior art limits its large-scale industrial application, especially since inhibitors in lignocellulose hydrolysate do not produce bacterial cellulose.
The bacterial cellulose was produced by static fermentation method using Komagataeibacter xylinus, which is resistant to lignocellulose hydrolysate inhibitors, and the inexpensive and easy-to-get lignocellulose hydrolysate as the carbon source.
The high yield of bacterial cellulose is achieved, reaching 5.5g/L, reducing production costs, and reducing environmental pollution and health hazards through green production processes.
Smart Images
Figure CN119931871A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial engineering, in particular to a xyloglucanacetobacter strain capable of producing high-yield bacterial cellulose in lignocellulose hydrolysate and its application. Background Art
[0002] Bacterial cellulose is an extracellular polysaccharide secreted by different species of bacteria. It is considered to be an environmentally friendly biomaterial that does not cause ecological pollution. It is also biodegradable and has excellent adhesion, skin elasticity, softness and moisturizing effects.
[0003] Although bacterial cellulose, as a promising biodegradable nanomaterial, has received significant attention from academia and industry in various research fields due to its excellent properties, its large-scale industrial application is still limited due to low productivity and high production cost. The key factor to solve the economic feasibility is to reduce the cost of raw materials, especially the cost of carbon sources. In order to achieve favorable yields and controlled costs, it has become a hot issue to find new and efficient raw materials or carbon sources from agricultural waste or industrial by-products to increase the yield of bacterial cellulose. The lignocellulosic hydrolysate prepared from agricultural waste or industrial by-products is rich in sugars and can be used for further fermentation to maximize the utilization of agricultural waste. However, some inhibitors contained in the lignocellulosic hydrolysate are not conducive to the production of bacterial cellulose. Therefore, using lignocellulosic hydrolysate as a suitable carbon source for culturing inhibitor-tolerant Bacillus xylosus can save carbon source costs and achieve green production. Summary of the invention
[0004] The present invention provides a strain of Komagataeibacter xylinus and a method for producing bacterial cellulose by fermenting a lignocellulose hydrolysate. The method uses a lignocellulose hydrolysate with a wide source and low price as a raw material, adds a suitable nitrogen source, and performs production in a static fermentation mode. The obtained product contains a large amount of bacterial cellulose, is suitable for the development of medical and cosmetic products, can be used for the harmless treatment of industrial wastewater, can effectively reduce the pollution of inhibitors such as furfural to the environment and the harm to human health, provides green, safe and efficient degradation and reuse of lignocellulose hydrolysate and bacterial cellulose production methods for related enterprises, and can greatly reduce the production cost of bacterial cellulose.
[0005] One of the technical solutions provided by the present invention is a strain of Komagataeibacter xylinus, specifically Komagataeibacter xylinus, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 9, 2023, with a strain deposit number of CGMCC NO.27300, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The Komagataeibacter xylinus is obtained by adaptive training of Komagataeibacter xylinus CGMCC 2955. After adaptive training, Komagataeibacter xylinus is tolerant to inhibitors such as furfural and acetic acid, and can use lignocellulose hydrolysate as a carbon source to ferment and produce bacterial cellulose, with a yield of 5.5 g / L.
[0006] The second technical solution provided by the present invention is the use of Komagataeibacter xylinus in the production of bacterial cellulose by fermentation of lignocellulose hydrolysate; Furthermore, the method for producing bacterial cellulose by fermentation of Komagataeibacter xylinus is as follows: A single colony of the experimental engineering bacteria was inoculated into a 100 mL shake flask containing 30 mL of seed culture medium, and cultured in a shaker at 30°C and 200 rpm for 48 h to prepare a first-level seed solution; at an inoculum rate of 1%, it was inoculated into a 250 mL shake flask containing 100 mL of seed culture medium, and cultured in a shaker at 30°C and 200 rpm for 48 h to prepare a second-level seed solution; The secondary seed liquid was inoculated at 3-5% into a fermentation medium with lignocellulose hydrolyzate as a carbon source for static fermentation at a temperature of 30°C for 7 days, and the bacterial cellulose yield reached 5.5 g / L. Furthermore, the culture medium used for seed culture is: 2.5% (w / v) glucose, 0.75% (w / v) yeast powder, 1% (w / v) peptone, 1% (w / v) disodium hydrogen phosphate dodecahydrate, and the rest is water, pH 6; Furthermore, the fermentation medium used in static fermentation is: 50% (v / v) lignocellulose hydrolysate, 0.75% (w / v) yeast powder, 1% (w / v) peptone, 1% (w / v) disodium hydrogen phosphate dodecahydrate, and the rest is water, pH 6; Furthermore, the preparation method of the lignocellulose hydrolysate is as follows: 20-50 mesh activated carbon powder and lignin hydrolysate are mixed at a solid-liquid ratio of 1:10, and treated in a shaking incubator at 30°C and 200 rpm for 3 hours. The precipitate is removed by centrifugation, and the pH is 6.
[0007] The effects of the present invention are: The present invention is a method for producing bacterial cellulose by static fermentation under certain temperature conditions using Bacillus xylosus that tolerates lignocellulose hydrolysate inhibitors as strains, lignocellulose hydrolysate as raw materials, supplemented with yeast powder and peptone as carbon sources. Compared with the prior art, the present invention has the following advantages: phenolic and aldehyde inhibitors contained in lignocellulose hydrolysate will inhibit the growth of Bacillus xylosus and the generation of fermentation products, and ultimately affect the yield of bacterial cellulose. The present invention uses a strain of Bacillus xylosus that tolerates lignocellulose hydrolysate inhibitors and has a high bacterial cellulose yield as a fermentation strain, and the strain is safe and stable; the present invention uses cheap, easily available, and widely available lignocellulose hydrolysate as a fermentation raw material, and adopts a biological fermentation method to increase the recycling of agricultural by-products; the raw materials used in the present invention are easy to obtain, and bacterial cellulose can be obtained by fermentation; the static fermentation medium used in the present invention has a simple formula, controllable quality, and low cost. After optimization, suitable nutrients such as nitrogen sources are added to promote the growth and production of microorganisms, and can be used for large-scale industrial production; the present invention adopts a green production process, a simple operation process, and no wastewater or waste discharge, so as to achieve green production of bacterial cellulose. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 The wet film state of the bacterial cellulose after purification and the dry film state after pressing and drying in Example 1 of the present invention; DETAILED DESCRIPTION In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.
[0009] The fermentation strain used in the embodiment of the present invention is Komagataeibacter xylinus, and the strain collection number is CGMCC NO.27300.
[0010] The present invention is further explained below through specific implementation modes.
[0011] Example 1: Method for producing bacterial cellulose by static fermentation of lignin hydrolysate by Komagataeibacter xylinus (1) Preparation of seed culture medium: 2.5% (w / v) glucose, 0.75% (w / v) yeast powder, 1% (w / v) peptone, 1% (w / v) disodium hydrogen phosphate dodecahydrate, and the remainder water. Adjust the pH to 6 with glacial acetic acid and sterilize at 115°C for 30 min.
[0012] (2) Preparation of lignocellulose hydrolysate: 20-50 mesh activated carbon powder was mixed with lignocellulose hydrolysate at a solid-liquid ratio of 1:10 and treated in an oscillating incubator at 30°C and 200 rpm for 3 h. After centrifugation to remove the precipitate, the pH was adjusted to 6 using NAOH and sterilized at 115°C for 30 min. (2) Preparation of fermentation medium: 50% (v / v) lignin hydrolyzate, 0.75% (w / v) yeast powder, 1% (w / v) peptone, 1% (w / v) disodium hydrogen phosphate dodecahydrate, and the rest water. The pH was adjusted to 6 with NaOH and sterilized at 115°C for 30 min.
[0013] (3) Preparation of primary seed solution: Under sterile conditions, inoculate a ring of Xylella fastidiosa into the seed culture medium and culture it in a shaking incubator at 30°C and 200 r / min for 48 h. (4) Preparation of secondary seed liquid: Under sterile conditions, the bacterial cellulose pellets were squeezed and discarded, and the remaining fermentation liquid was inoculated into the seed culture medium at a rate of 1% of the primary seed liquid, and cultured in a shaking incubator at 30°C and 200 r / min for 48 h. (5) Fermentation: Under sterile conditions, the bacterial cellulose pellets were squeezed and discarded, and the remaining fermentation liquid was inoculated into the fermentation medium at a 3% inoculation rate with the secondary seed liquid. The fermentation temperature was 30°C and the static culture was carried out for 7 days.
[0014] (6) Purification of bacterial cellulose: After fermentation, the bacterial cellulose membrane was immersed in 1 M NaOH solution until the membrane turned white, and then washed with distilled water to pH 7.
[0015] (7) Detection of bacterial cellulose yield: The purified bacterial cellulose membrane was pressed into a constant weight dry sheet using a sheet machine, stored and weighed, and the bacterial cellulose yield was measured to be 5.5 g / L.
Claims
1. A strain of Gluconobacter xylinum for efficiently utilizing lignocellulose hydrolysate to ferment and produce bacterial cellulose and its application, characterized in that: The Komagataeibacter xylinus is obtained by adaptive domestication from a high-yield bacterial cellulose strain as a starting strain. The preservation number of the high-yield bacterial cellulose strain obtained by adaptive domestication is CGMCC NO.27300.
2. The use according to claim 1, characterized in that The method for producing bacterial cellulose by fermentation is as follows: a single colony of the experimental engineering bacteria is inoculated into a 100 mL shake flask containing 30 mL of seed culture medium, and cultured in a shaker at 30°C and 200 rpm for 48 hours to prepare a first-level seed solution; with an inoculum of 1%, it is inoculated into a 250 mL shake flask containing 100 mL of seed culture medium, and cultured in a shaker at 30°C and 200 rpm for 48 hours to prepare a second-level seed solution. The inoculum of 3% is transferred to a 100 mL shake flask with a liquid volume of 100 mL. The fermentation temperature is 30°C, and the fermentation is static.