Method for increasing yield of bacterial cellulose by using blue-green algae

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a method for increasing the yield of bacterial cellulose through blue-green algae. Comprising the following steps: carrying out fermentation by adopting Komagataeibacter xylinus to produce bacterial cellulose, and adding blue-green algae in the fermentation process; the ratio of the total viable count of the gluconacetobacter xylinum to the total viable count of the blue-green algae is (0.3-0.5): 1. According to the research, the problem that a compact film is formed on the surface of a culture medium by primarily generated bacterial cellulose can be remarkably solved by adding the blue-green algae (especially synechocystis) into a gluconacetobacter xylinum fermentation system, the cellulose production efficiency is improved, and the method has important value in the field of fermentation production of the bacterial cellulose.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation engineering, and particularly relates to a method for improving the yield of bacterial cellulose by using cyanobacteria. BACKGROUND

[0002] Bacterial cellulose is an extracellular polysaccharide produced by microorganisms through fermentation. It is a high-purity and high-crystallinity cellulose with unique physical and chemical properties. Compared with plant cellulose, bacterial cellulose has a finer nanofiber diameter (usually 20-100 nanometers) and higher mechanical strength, good biocompatibility and biodegradability. These characteristics make bacterial cellulose have broad application prospects in many fields. Bacterial cellulose can be used to manufacture biomedical materials, such as dressings for skin tissue repair; can be applied to the food industry, such as as a food additive to improve the taste of beverages; and can be applied to the field of material science, such as in the preparation of flexible electronic devices.

[0003] The production industry of bacterial cellulose has developed rapidly in recent years. At present, the production methods mainly include static culture method and dynamic culture method. The static culture method is simple to operate, but the production efficiency is low and the cellulose yield is limited. The dynamic culture method, such as stirred tank fermentation, can improve the mass and heat transfer efficiency, but there are problems such as reduction of bacterial cellulose yield and quality, and reduction of strain stability. Gluconacetobacter xylinus is one of the main strains for producing bacterial cellulose. It is a gram-negative bacterium with good cellulose synthesis capacity. This strain can grow in a culture medium containing carbon source (such as glucose) and nitrogen source, and synthesize cellulose from glucose through its cell surface cellulose synthase complex. The cellulose synthesized by Gluconacetobacter xylinus has high crystallinity and purity, and the cellulose nanofibers form a dense network structure through hydrogen bonding interaction.

[0004] Although there is a method for producing bacterial cellulose by static fermentation of Gluconacetobacter xylinus at present, the effect of improving cellulose yield by optimizing fermentation conditions and strain modification is limited, and the yield of bacterial cellulose needs to be further improved to meet the needs of large-scale industrial application. SUMMARY

[0005] In order to solve the problems existing in the prior art, the present application provides a method for improving the yield of bacterial cellulose by using cyanobacteria.

[0006] In a first aspect, the present application provides a method for improving the yield of bacterial cellulose, comprising: adopting Gluconacetobacter xylinus to produce bacterial cellulose by fermentation, and adding cyanobacteria in the process of fermentation; the total viable bacteria ratio of the Gluconacetobacter xylinus and the cyanobacteria is (0.3-0.5):1.

[0007] The Gluconacetobacter xylinus generally adopts static fermentation in the production of bacterial cellulose. When static culture is adopted, the produced bacterial cellulose is in the form of film, which is beneficial to further application. However, if dynamic fermentation is adopted, the bacterial cellulose can only be in the form of granules. However, the bacterial cellulose film produced by static fermentation is formed on the surface of the culture medium, which leads to insufficient oxygen supply and inhibits the further production of cellulose. In order to solve this problem, the prior art adopts the method of shaking first and then static culture for the fermentation production of bacterial cellulose. However, this method only increases the amount of Gluconacetobacter xylinus, and still cannot effectively solve the problem of yield reduction caused by the formation of bacterial cellulose film on the surface of the culture medium. In addition, in order to maintain the integrity of the structure of the bacterial cellulose film, oxygen cannot be directly introduced. The present application aims at these problems. The cyanobacteria is added to the fermentation system of Gluconacetobacter xylinus. It is found in the actual experiment process that the two have good compatibility and do not have antagonistic effect on the production of cellulose. They can grow together under the conditions suitable for the production of bacterial cellulose. At the same time, the cyanobacteria can generate oxygen by using the CO2 produced by the fermentation of Gluconacetobacter xylinus, which can alleviate the problem of insufficient oxygen supply caused by the formation of bacterial cellulose film on the surface of the culture medium. This effectively solves the problem of low yield of bacterial cellulose of Gluconacetobacter xylinus under the condition of static fermentation.

[0008] In some specific embodiments of the present application, the inoculation amount of the Gluconacetobacter xylinus is 1×10 5~10 cfu / mL.

[0009] Further, the fermentation conditions include: static fermentation culture at 28-35℃.

[0010] Further, the culture medium used in the fermentation process includes, in parts by weight: 0.5-2 parts of yeast powder, 0.5-3 parts of peptone, 0.5-3 parts of disodium hydrogen phosphate dodecahydrate, and 1.5-4 parts of glucose.

[0011] Further, the cyanobacteria includes one or more of Microcystis, Anabaena, Synechococcus, Nostoc, Oscillatoria, Spirulina, Chroococcus, Chlamydomonas, Chroomonas, Chroococcidiopsis, or Synechocystis.

[0012] Further, the cyanobacteria is Synechocystis.

[0013] In the second aspect, the present application provides a bacterial cellulose film, which is prepared by the above-mentioned method.

[0014] In the third aspect, the present application provides a mutualistic microorganism combination, which includes Gluconacetobacter xylinus and cyanobacteria; and the cyanobacteria is preferably Synechocystis.

[0015] Further, the ratio of the total viable cell count of the Gluconacetobacter and the cyanobacterium is (0.3-0.5):1.

[0016] In a fourth aspect, the present application provides use of the aforementioned mutualistic microorganism combination in improving the yield of bacterial cellulose.

[0017] The present application has the following advantages: The present application adds cyanobacterium (especially Synechococcus) to the fermentation system of Gluconacetobacter, and the two can be well compatible, grow under suitable conditions for the fermentation production of bacterial cellulose, and at the same time can overcome the problem of the decrease of the yield of bacterial cellulose after the bacterial cellulose is formed on the surface of the culture medium.

[0018] The method provided by the present application can significantly improve the yield of bacterial cellulose, and is beneficial to the large-scale industrial production of bacterial cellulose. DETAILED DESCRIPTION

[0019] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application are described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0020] In the following examples, the experimental methods involved, if not specifically mentioned, are all conventional methods in the art, for example, can refer to the experimental manuals in the art, or follow the conditions suggested in the manufacturer's instructions.

[0021] In the following examples, the experimental materials and reagents involved, if not specifically mentioned, can be obtained from commercial channels.

[0022] The culture medium used in the following examples is as follows (wt%): HS liquid culture medium: 0.75% yeast powder, 1% peptone, 1% disodium hydrogen phosphate dodecahydrate, 2.5% glucose, dissolved in deionized water.

[0023] When configuring, 0.75% yeast powder, 1% peptone and 1% disodium hydrogen phosphate dodecahydrate are dissolved in water, and after mixing, the pH of the culture medium is adjusted to 4.5 by phosphoric acid; 2.5% glucose is dissolved in water, and after sterilization, the two are combined to obtain the culture medium.

[0024] HS solid culture medium: 0.75% yeast powder, 1% peptone, 1% disodium hydrogen phosphate dodecahydrate, 2.5% glucose, 1.5% agar powder, and the pH of the culture medium is adjusted to 4.5 by phosphoric acid.

[0025] BG11 medium: NaNO3 1.5 g / L, K2HPO4 40 mg / L, MgSO4 36.6 mg / L, CaCl2 27.2 mg / L, Citric acid 6 mg / L, Ammonium ferric citrate 6 mg / L, EDTA Na 21 mg / L, Na2CO3 20 mg / L, H3BO3 2.86 mg / L, MnCl2·4H2O 1.81 mg / L, ZnSO4 0.22 mg / L, Na2MoO4·2H2O 0.39 mg / L, CuSO4·5H2O 0.08 mg / L, CoCl2·6H2O 0.0409 mg / L, dissolved in deionized water.

[0026] BG11 solid medium: NaNO3 1.5 g / L, K2HPO4 40 mg / L, MgSO4 36.6 mg / L, CaCl2 27.2 mg / L, Citric acid 6 mg / L, Ammonium ferric citrate 6 mg / L, EDTA Na 21 mg / L, Na2CO3 20 mg / L, H3BO3 2.86 mg / L, MnCl2·4H2O 1.81 mg / L, ZnSO4 0.22 mg / L, Na2MoO4·2H2O 0.39 mg / L, CuSO4·5H2O 0.08 mg / L, CoCl2·6H2O 0.0409 mg / L, agar powder 1.5% g / L.

[0027] The Gluconacetobacter in the following examples is Gluconacetobacter xylinus KxPTS3, which has been disclosed in patent CN202311738547.7.

[0028] Example 1 1. Gluconacetobacter was streaked on HS solid medium containing 50 μg / mL of kanamycin resistance and activated, the culture temperature was 30°C, and the culture time was 48 hours; Synechocystis was streaked on BG11 solid medium and activated, 15 μmol m -2 s -1 Light intensity photoperiod culture, culture temperature 30°C, culture time 4d; 2. The solid-cultured Gluconacetobacter was transferred to 20 mL of HS liquid medium supplemented with 50 μg / mL of kanamycin resistance for expansion culture as a primary seed liquid, the culture temperature was 30°C, the shaking speed was 200 rpm, and the culture time was 48h; the solid-cultured Synechocystis was transferred to 200 mL of BG11 liquid medium for expansion, 15 μmol m -2 s -1Light intensity light culture, culture temperature 30℃, shaking speed 200rpm, culture time 4d; 3、The bacteria cellulose in the primary seed liquid of Gluconacetobacter xylinus was broken and transferred to 70 mL of HS liquid medium with 50ug / mL of kanamycin resistance for secondary seed liquid culture, culture temperature 30℃, shaking speed 200rpm, culture time 48h; 4、The bacteria cellulose in the secondary seed liquid of Gluconacetobacter xylinus was broken and transferred to 750mL of HS liquid medium with 50ug / mL of kanamycin resistance, and Synechocystis was collected by centrifugation and added to the bacteria cellulose fermentation system, 15 μmol m -2 s -1 Light intensity light culture, culture temperature 30℃, shaking speed 200rpm, culture time 4d; The active bacteria concentration of Gluconacetobacter xylinus and Synechocystis transferred into the bacteria cellulose fermentation system was calculated by plate dilution counting method. In this embodiment, the volume of Gluconacetobacter xylinus transferred into the bacteria cellulose fermentation system was 70mL, and the active bacteria concentration was about 6.61×10 8 cfu / mL. The volume of Synechocystis transferred into the bacteria cellulose fermentation system was 200mL, and the active bacteria concentration was about 4.96×10 8 cfu / mL. The active bacteria ratio of Gluconacetobacter xylinus and Synechocystis was 0.47:1.

[0029] The results were statistically analyzed, and the yield of bacteria cellulose was 7.48 g / L.

[0030] Example 2 The method of this embodiment is the same as that of Example 1, except that: The volume of Gluconacetobacter xylinus transferred into the bacteria cellulose fermentation system was 70mL, and the active bacteria concentration was about 6.96×10 8 cfu / mL.

[0031] The volume of Synechocystis transferred into the bacteria cellulose fermentation system was 200mL, and the active bacteria concentration was about 5.36×10 8 cfu / mL. The active bacteria ratio of Gluconacetobacter xylinus and Synechocystis was 0.45:1.

[0032] The results were statistically analyzed, and the yield of bacteria cellulose was 7.18g / L.

[0033] Example 3 The method of this embodiment is the same as that of Example 1, except that: The volume of Gluconacetobacter xylinus transferred into the bacteria cellulose fermentation system was 70mL, and the active bacteria concentration was about 6.82×10 8 cfu / mL.

[0034] The volume of Synechocystis sp. introduced into the bacterial cellulose fermentation system was 200 mL, and the viable cell concentration was about 3.68 x 10 8 The viable cell ratio of Gluconacetobacter and Synechocystis sp. was 0.65:1.

[0035] The results were statistically analyzed, and the yield of bacterial cellulose was 5.21 g / L.

[0036] Example 4 The method of this example was the same as that of Example 1, except that: The volume of Gluconacetobacter introduced into the bacterial cellulose fermentation system was 70 mL, and the viable cell concentration was about 5.63 x 10 8 cfu / mL.

[0037] The volume of Synechocystis sp. introduced into the bacterial cellulose fermentation system was 200 mL, and the viable cell concentration was about 6.0 x 10 8 cfu / mL. The viable cell ratio of Gluconacetobacter and Synechocystis sp. was 0.33:1.

[0038] The results were statistically analyzed, and the yield of bacterial cellulose was 6.5 g / L.

[0039] Example 5 The method of this example was the same as that of Example 1, except that: The volume of Gluconacetobacter introduced into the bacterial cellulose fermentation system was 70 mL, and the viable cell concentration was about 6.56 x 10 8 cfu / mL.

[0040] The volume of Synechocystis sp. introduced into the bacterial cellulose fermentation system was 200 mL, and the viable cell concentration was about 6.96 x 10 8 cfu / mL. The viable cell ratio of Gluconacetobacter and Synechocystis sp. was 0.33:1.

[0041] The results were statistically analyzed, and the yield of bacterial cellulose was 6.42 g / L.

[0042] Example 6 The method of this example was the same as that of Example 1, except that: The volume of Gluconacetobacter introduced into the bacterial cellulose fermentation system was 70 mL, and the viable cell concentration was about 8.03 x 10 8 cfu / mL.

[0043] The volume of Synechocystis sp. introduced into the bacterial cellulose fermentation system was 100 mL, and the viable cell concentration was about 4.96 x 10 8 cfu / mL. The viable cell ratio of Gluconacetobacter and Synechocystis sp. was 1.13:1.

[0044] The results were statistically analyzed, and the yield of bacterial cellulose was 5.45 g / L.

[0045] Example 7 The method of this example was the same as that of Example 1, except that: The volume of Gluconacetobacter bacteria introduced into the bacterial cellulose fermentation system was 70 mL, and the viable bacterial concentration was about 6.45 x 10 8 cfu / mL.

[0046] The volume of Synechococcus bacteria introduced into the bacterial cellulose fermentation system was 300 mL, and the viable bacterial concentration was about 5.92 x 10 8 cfu / mL. The ratio of viable Gluconacetobacter bacteria to Synechococcus bacteria was 0.25:1.

[0047] The results were statistically analyzed, and the yield of bacterial cellulose was 5.23 g / L.

[0048] Example 8 The method of this example was the same as that of Example 1, except that: 8 The volume of Gluconacetobacter bacteria was 70 mL, and the viable bacterial concentration was about 6.64 x 10 8 cfu / mL; Synechococcus bacteria was replaced by Anabaena, and the addition amount was 200 mL, and the viable bacterial concentration was about 5.28 x 10 8 cfu / mL, and the ratio of viable bacteria was 0.44:1.

[0049] The yield of bacterial cellulose was 5.65 g / L.

[0050] Comparative Example 1 The method of this comparative example was the same as that of Example 1, except that: The volume of Gluconacetobacter bacteria introduced into the bacterial cellulose fermentation system was 70 mL, and the viable bacterial concentration was about 6.45 x 10 8 cfu / mL.

[0051] Synechococcus bacteria was not introduced.

[0052] The results were statistically analyzed, and the yield of bacterial cellulose was 5.87 g / L.

[0053] From the results of Examples 1-8 and Comparative Example 1 above, it can be seen that when Synechococcus bacteria was not added, the yield was 5.87 g / L, and after adding Synechococcus bacteria at different ratios, the yield of bacterial cellulose was significantly improved within the viable bacterial ratio range of (0.33-0.47):1, and the highest yield reached 7.48 g / L, which increased by 27.4%. However, when the viable bacterial ratio was 0.25:1 or 0.65:1 or more, the yield of bacterial cellulose decreased significantly.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for increasing bacterial cellulose production, characterized in that: include: Bacterial cellulose is produced by fermentation using Gluconacetobacter xylans, and cyanobacteria are added during the fermentation process; the ratio of the total viable bacteria count of the Gluconacetobacter xylans to the total viable bacteria count of the cyanobacteria is (0.3-0.5):

1.

2. The method according to claim 1, characterized in that The fermentation conditions include: Static fermentation culture was carried out at 28~35℃.

3. The method according to claim 1 or 2, characterized in that The culture medium used in the fermentation process includes, by weight, 0.5-2 parts of yeast powder, 0.5-3 parts of peptone, 0.5-3 parts of disodium hydrogen phosphate dodecahydrate, and 1.5-4 parts of glucose.

4. The method according to claim 1, wherein The cyanobacteria include one or more of Microcystis, Anabaena, Aphanizomenon, Nostoc, Oscillatoria, Spirulina, Glechoma, Planospora, Cryptophyta, Chrococcus or Synechocystis.

5. The method according to claim 4, characterized in that The cyanobacteria is Synechocystis.

6. A bacterial cellulose membrane, characterized in that The bacterial cellulose membrane is prepared by the method according to any one of claims 1 to 5.

7. A mutualistic microbial combination, characterized in that: include: Gluconacetobacter xylosarcina and cyanobacteria; the cyanobacteria are preferably Synechocystis sp.

8. The symbiotic microbial combination according to claim 7, characterized in that: The ratio of the total viable bacteria count of the xylella fastidiosa to the total viable bacteria count of the cyanobacterium is (0.3-0.5):

1.

9. Use of the symbiotic microbial combination according to claim 7 or 8 in increasing bacterial cellulose production.