KR100530996B1 - Method for Producing Bacterial Cellulose Using the Waste of Beer Fermentation Broth - Google Patents

Abstract

The present invention relates to a method for producing microbial cellulose, wherein the microorganisms having the ability to produce microbial cellulose are cultured in beer fermentation waste yeast.

According to the present invention, when the beer fermentation waste yeast solution is used, the production of microbial cellulose is reduced by inducing the production of microbial cellulose in the form of microbial cellulose by inhibiting the generation of mutant (Cel ^- mutant) that does not produce cellulose. It is possible to maximize efficiency and to reduce the cost of cellulose production.

Description

Method for Producing Bacterial Cellulose Using Beer Fermentation Waste Yeast Liquid {Method for Producing Bacterial Cellulose Using the Waste of Beer Fermentation Broth}

The present invention relates to a method for producing microbial cellulose, which comprises culturing microorganisms producing microbial cellulose in a fermentation broth.

Microorganisms capable of producing microbial cellulose are reported in the genus Acetobacter, Agrobacterium, Rhizobium, Pseudomonas, and Sarcina. Among them, the acetobacter genus is able to produce a large amount of cellulose secreted as extracellular fibrils rather than cell wall polymers found in eukaryotes.

The microbial cellulose produced by the genus Acetobacter has become a very important topic in the high value-added new material industry as well as food due to the unique characteristics not found in plant-derived cellulose. Since microbial cellulose can be mass-produced for a short period of time and have high fiber crystallinity, water retention, moldability, and tensile strength, it is being developed for various industrial purposes. Recently, it is used as a cosmetic pad, medical pad, enzyme immobilization carrier, paper coating, etc. have. In particular, US Patent No. 5274199 and 4912049 disclose a method for manufacturing acoustic diaphragm and artificial skin using microbial cellulose, respectively, and Korean Patent No. 2003-0015399 and 1997-0014612 describe dietary fiber and dietary fiber using microbial cellulose, respectively. A method for preparing a beverage is disclosed.

As described above, microbial cellulose can be used in various industrial materials, food materials, etc. due to its excellent physical properties, and in particular, it is an environmentally friendly material, which has infinite development potential and variety.

However, since cellulose produced by microorganisms has a relatively low production yield and high cost, the application of microbial cellulose has been limited to high value-added products such as speaker diaphragms.

In addition, agitation culture is more economical than stationary culture to produce microbial cellulose, but in general, microbial cellulose producing strains cannot produce cellulose due to shear stress generated in the incubator when shaken or stirred culture is performed. Cel ^- mutant occurs, and this mutant strain is faster than microbial cellulose producing strains, so in continuous aeration agitation, producers are culled and non-producers become the subjects of culture (Valla, S. and Kjosbakken, J., J. General Microb. 128: 1401-8, 1981) produced microbial cellulose by means of stationary culture, which has a very low productivity but requires a long incubation time and a lot of labor.

Therefore, even in agitated culture conditions, development of culture conditions that do not generate or reduce the frequency of occurrence of the aforementioned mutants (Cel ^- mutant) and development of a medium substitute material that can reduce the production cost of microbial cellulose are urgently needed. .

On the other hand, a method of producing microbial cellulose by stirring and acetobacter xylanum in a medium containing peptone, yeast extract, glucose, citric acid, ethanol and the like is known, but this is a cellulose production medium using expensive peptone, yeast extract, etc. Therefore, there is a disadvantage of low economic feasibility (Korean Patent Publication 1998-067009).

Accordingly, the present inventors have made a diligent effort to develop a method for economically producing microbial cellulose without the generation of mutants (Cel ^- mutants) that do not produce cellulose even under shear stress culture conditions, using waste yeast fermentation broth as a culture medium. As a result, it was confirmed that the production of microbial cellulose at high yield was possible, and the present invention was completed.

An object of the present invention is to provide a method for producing microbial cellulose without the generation of mutants (Cel ^- mutant) that does not produce cellulose even under shear stress culture conditions.

Another object of the present invention is to provide a continuous method for producing microbial cellulose suitable for industrial production.

Still another object of the present invention is to provide a method for producing microbial cellulose using waste yeast liquid, which is an industrial waste of a beer factory, as a medium substitute.

The present invention to achieve the above object, acetonitrile bakteo (Acetobacter) in, gluconic acetonitrile bakteo (Gluconacetobacter) genus, Agrobacterium (Agrobacterium), A separation tank emptying (Rhizobium) in and Pseudomonas from the group consisting of genus (Pseudomonas) Provided is a method for producing microbial cellulose, wherein the microorganism having the selected ability to produce cellulose is cultured in a medium containing beer fermentation waste yeast liquid.

In the present invention, the microorganism may be characterized as Gluconacetobacter hansenii PJK (KCTC 10505BP), and the culture may be characterized by a continuous batch culture method.

The continuous batch culture may include the steps of: (a) inoculating the culture supernatant into beer fermentation waste yeast after pre-culture of microorganisms having cellulose production ability; (b) culturing the microorganisms in beer fermentation waste yeast to produce microbial cellulose in the form of tubers; (c) inoculating a portion of the culture supernatant with fresh beer fermented spent yeast solution; (d) producing microbial cellulose from the inoculated culture solution; And (e) repeating steps (c) and (d).

The present invention also provides a method for producing microbial cellulose, which comprises culturing Gluconacetobacter hansenii PJK (KCTC 10505BP) in a medium containing 0.5 to 15% (v / v) ethanol. do.

According to the present invention, by culturing the microorganisms in a medium containing ethanol, the production of microbial cellulose by inhibiting the generation of cellulose (Cel ^- mutant) that does not produce cellulose in the culture medium and producing a microbial cellulose in the form of a tube It is possible to maximize the efficiency of the separation and separation process. In addition, it is possible to maximize the productivity of microbial cellulose by performing a continuous culture process for producing microbial cellulose. In addition, it is possible to reduce the production cost by producing a microbial cellulose from the microorganism by using the supernatant of the waste yeast liquid which is an industrial waste of a beer factory as a medium.

In the present invention, when the ethanol concentration in the medium is less than 0.5% (v / v), there is a problem that it is difficult to suppress the generation of cellulose (Cel ^- mutant) that does not produce cellulose, 15% (v / v) If exceeded, there is a problem that the growth of microorganisms is lowered and the productivity is lowered.

The present invention also provides Gluconacetobacter hansenii PJK (KCTC 10505BP) having cellulose generating ability.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, a single microorganism capable of producing microbial cellulose was first isolated from a decaying apple, and a 13SB nucleotide sequence (SEQ ID NO: 1) was analyzed by 16S rDNA complete sequencing, followed by analysis of 16S rDNA similarity and phylogenetic tree. results the microorganisms were gluconate acetonitrile bakteo century you have been identified as (Gluconacetobacter hansenii), gluconate acetonitrile bakteo century (Gluconacetobacter hansenii) named PJK, and deposited at the Research Institute of Bioscience and biotechnology on August 11, 2003 (KCTC 10505BP) .

After incubation of the microorganisms, the culture supernatant was inoculated in a fresh medium containing 1% (v / v) ethanol and a fresh medium containing no ethanol, respectively, and shaken to incubate the microbial cellulose unproduced mutant strain under the shear stress environment. Frequency was checked. At this time, the incidence of microbial cellulose unproduced mutants was confirmed from the form of colonies obtained by culturing the culture solution on agar medium.

As a result, it was confirmed that the incidence of microorganisms grown in the ethanol-containing medium was reduced in microbial cellulose-producing mutants. In addition, it was confirmed that the microbial cellulose produced in the ethanol-containing medium was produced in the form of lumps for easy recovery and separation, and the yield of cellulose was also increased.

In addition, the supernatant of the culture solution was inoculated in a new medium containing ethanol and cultured, and this process was carried out continuously, and it was confirmed that a continuous production process of microbial cellulose was possible.

The culture of the microorganisms using the supernatant of the waste yeast liquor, an industrial waste of the beer factory, as a medium showed similar results to the productivity of the microbial cellulose produced in the microbial cellulose production medium. It was confirmed not to.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples only illustrate the present invention in detail and do not limit the present invention by these examples.

In particular, in the following examples, Gluconacetobacter hansenii PJK was used as the cellulose producing strain, but it will be apparent to those skilled in the art that the same result can be obtained using other cellulose producing strains.

Example 1 Gluconacetobacter Hanseni Gluconacetobacter hansenii Separation and Identification of PJK

To prepare a pH 5 medium solution of the composition as shown in the following table. After sterilizing the medium solution at 121 ° C. for 15 minutes, 50 mL was dispensed into a 250 mL Erlenmeyer flask, a portion of the decayed apple was added, and cultured at 30 ° C., followed by dilution of the culture solution in which cellulose pellicle was present in saline solution. It was plated on a solid medium of the same composition as 1 and incubated at 30 ° C. Colonies formed on a solid medium were repeated five times in this manner to isolate single bacteria capable of producing microbial cellulose.

ingredient Volume (/ L) Glucose (SIGMA, USA) Yeast Extract (DIFCO, USA) Peptone (DIFCO, USA) Sodium Phosphate (Pharmacological Chemistry, Japan) Citric Acid (Pharmacological Chemistry, Japan) Acetic Acid (Dongyang Chemical, Korea) Ethanol (Dongyang Chemical, Korea) Cycloheximide (SIGMA, USA) 20 g 5 g 5 g 2.7 g 1.15 g 2 mL 5 mL 0.1 g

Total sequence (1376 bp) of isolated single strain 16S rDNA was determined (Juke, TH and Cantor, CR, Evolution of protein molecules.In mammalian protein metabolism, Edited by HN Munro. P21. Academic Press, New York., 1969) Analysis of percent similarity and phylogenetic tree (Saito, N. and Nei, M., Mol. Biol. Evol. 4: 406-425 (1987)), as shown in Table 2 and Figure 1 Gluconacetobacter represented by the type strain of hansenii and high 16S rDNA similarity and 1000 high bootstrap value of 99.27% (bootstrap value) the microorganism was identified as gluconic acetonitrile bakteo century Needle (Gluconacetobacter hansenii), gluconate acetonitrile bakteo century Needle (Gluconacetobacter hansenii ) PJK.

Standard strain % Similarity Gluconacetobacter hansenii NCIMB 8746 (T) 99.27 Gluconacetobacter europaeus DSM 6160 (T) 98.40 Gluconacetobacter xylinus subsp.sucrofermentans BPR2001 98.33 Gluconacetobacter xylinus subsp.xylinus NCIMB 11664 (T) 98.25 Acetobacter intermedius DSM 11804 (T) 98.03 Acetobacter oboediens DSM 11826 (T) 98.03 Gluconacetobacter liquefaciens IFO 12388 (T) 96.72 Gluconacetobacter diazotrophicus ATCC 49037 (T) 96.58 Acetobacter aceti (JCM 7641 (T)) 94.61 Acetobacter pomorum DSM 11825 (T) 93.95 Acetobacter pasteurianus LMG 1262 (T) 93.80

The microorganism is a gram-negative bacillus, oxidizing ethanol, lactic acid, acetic acid (Park, JK et al., Biotechnol. Bioprocess Eng. 8: 83-88, 2003), and colonies formed in solid medium Has a rounded and rough surface.

Example 2: Gluconacetobacter Hanseni in a medium containing ethanol ( Gluconacetobacter hansenii Cultivation of PJK

To prepare a pH 5 medium solution of the composition shown in Table 3. After sterilizing the medium solution for 15 minutes at 121 ° C, dispense 50 mL into a 250 mL Erlenmeyer flask and inoculate Gluconacetobacter hansenii PJK strain, a microbial cellulose producing strain, at a speed of 200 rpm at 30 ° C. in a shake incubator. Pre-culture for 1 day while rotating. Subsequently, the supernatant of the preculture solution was inoculated in a 50 mL fresh medium solution containing 1% (v / v) ethanol in 5 mL (v / v) in a 250 mL Erlenmeyer flask and shaken at 30 ° C. at a speed of 200 rpm for 5 days. Incubated.

ingredient Volume (/ L) Glucose (SIGMA, USA) Yeast extract (DIFCO, USA) Peptone (DIFCO, USA) Succinic acid (WACO PURE CHEMICAL, USA) Acetic acid (Dongyang Chemical, Korea) 10 g 10 g 7 g 0.2 g 1.5 mL

After incubation, the culture was recovered and centrifuged at 4000 rpm for 20 minutes. Thereafter, the supernatant was removed, washed twice with distilled water and centrifuged in the same manner as described above, and frozen at minus 50 ° C. until a constant volume was obtained to determine the dry weight of the microbial cellulose containing the cells. Thereafter, 20 mL of 0.3N sodium hydroxide solution was added to the cellulose containing the cells, followed by boiling for 5 minutes to dissolve all the cells. The pure microbial cellulose from which the cells were removed was sufficiently washed until neutral and lyophilized again to measure dry weight. The dry weight of the cells was measured by the difference between the dry weight of the microbial cellulose containing the cells and the dry weight of the pure microbial cellulose.

As a result of the measurement, the dry weights of the cells and microbial cellulose in the ethanol-containing medium showed 3.34 g / L and 2.31 g / L, respectively.

Example 3: Microbial Cellulose Production in Tube Form by Ethanol-Containing Medium

The form of the microbial cellulose produced in Example 2 is shown in FIG. 2. The microbial cellulose produced in the medium containing ethanol began to form small lumps of small spherical cellulose pellets on the second day of cultivation. After enclosing the whole mass, the film gradually thickened to form one large mass as shown in FIG. 2.

Example 4: Mutant strains that do not produce microbial cellulose in a medium containing ethanol (Cel ^-  mutant occurrence

In the same manner as in Example 2, the supernatant of the preculture solution was inoculated with 5% (v / v) in 50 mL of a medium containing 1% (v / v) ethanol, incubated at 30 ° C. and 200 rpm, and then taken daily. The culture supernatant was diluted 10 ⁵ times in saline and then plated on a solid medium having the composition shown in Table 1 and incubated at 30 ° C. for 2 days.

The incidence of mutant strains that do not produce microbial cellulose from the form of colonies produced on solid media (Cel ^- mutant colony number / total colony number) was measured and the results are shown in Table 4 below. Cellulose-producing strains in solid media form rough-type colonies, and non-cellulose-forming strains form smooth-type colonies (Valla, S. and Kjosbakken, J., J. General Microb. 128: 1401-8, 1981).

Incubation time (day) Total viable count (CFU / mL) Frequency of mutant strain (Cel ^- mutant / total cell) One 7.7 × 10 ⁶ 0.18 2 7.8 × 10 ⁶ 0.13 3 2.57 × 10 ⁷ 0.004 4 6.2 × 10 ⁶ 0.0 5 4.32 × 10 ⁷ 0.002

As shown in Table 4, the microorganisms cultured in a medium containing 1% (v / v) ethanol was found that almost no mutant strain that does not produce microbial cellulose after 3 days of culture.

Example 5: Microbial Cellulose Production Using Continuous Batch Culture in Medium Containing Ethanol

After incubation in the same manner as in Example 2, the culture supernatant was inoculated with 5% (v / v) in 50 mL of a medium containing 1% (v / v) ethanol and the process shown in FIG. 3 at 30 ° C. and 200 rpm. Continuous batch culture was performed. Table 5 shows the amount of microbial cellulose produced after incubation for 5 days.

Incubation time (day) Dry weight of microbial cellulose (g / L) Number of batch cultures 1 time Episode 2 3rd time 4 times 5 times One 2.14 0.94 0.74 0.66 0.58 3 2.13 1.34 1.49 2.07 1.72 5 2.27 1.66 1.74 0.36 0.40

As shown in Table 5, when continuous batch culture of the microorganisms cultured for 3 days in a medium containing 1% (v / v) ethanol, it was found that the microbial cellulose production capacity is maintained up to 5 times.

Example 6: Composition Analysis of Beer Fermentation Waste Yeast Liquid

After centrifugation of the beer fermented waste yeast liquid at 4000 rpm, the supernatant was collected and sterilized at 121 ° C. for 15 minutes, and analyzed by gas chromatography. The beer fermented waste yeast solution contained 8.3% (v) of ethanol and acetic acid, respectively. / v) and 6.9% (v / v), glucose was included at a concentration of 0.28g / L, and the dry weight of the solid was 122g / L.

Example 7: Gluconacetobacter Hanseni using beer fermented waste yeast as a medium ( Gluconacetobacter hansenii  Cultivation of PJK

After centrifugation of the beer fermentation waste yeast liquid at 4000rpm, the supernatant was collected, and 50mL was put into a 250mL Erlenmeyer flask and sterilized at 121 ° C for 15 minutes.

After the pre-cultivation by the method of Example 2 5% (v / v) of the culture supernatant was inoculated into the supernatant of the beer fermentation waste yeast liquid and shaken at 200rpm, 30 ℃. After cultivation, samples were taken daily to measure the production of microbial cellulose in the same manner as in Example 2 (FIG. 4), and the incidence of mutants (Cel ^- mutant) not producing cellulose was measured in the same manner as in Example 4. (Table 6).

Incubation time (day) Total viable count (CFU / mL) Frequency of mutant strain (Cel ^- mutant / total cell) One 5.3 × 10 ⁶ 0.019 2 1.2 × 10 ⁷ 0.008 3 4.1 × 10 ⁶ 0 4 3.8 × 10 ⁶ 0.079 5 5.8 × 10 ⁶ 0 6 2.4 × 10 ⁶ 0 7 1.1 × 10 ⁶ 0 8 6.1 × 10 ⁶ 0 10 7.5 × 10 ⁶ 0 11 6.3 × 10 ⁶ 0 13 5.0 × 10 ⁶ 0 14 1.4 × 10 ⁷ 0 16 1.2 × 10 ⁷ 0

As shown in FIG. 4, it was found that the microbial cellulose production amount was 1.37 g / L at 16 days of culture, which was almost similar to the production amount of cellulose (Comparative Example 1) produced from the cellulose production medium containing no ethanol. In Figure 4 ● indicates the dry weight of cellulose, ▲ indicates the pH, ■ indicates the glucose concentration.

In addition, as shown in Table 6, it was found that almost no mutant (Cel ^- mutant) that does not produce cellulose.

Comparative Example 1: Gluconacetobacter Hanseni in a medium without ethanol ( Gluconacetobacter hansenii Cultivation of PJK

After pre-culture of the Gluconacetobacter hansenii PJK strain in the same manner as in Example 2, the culture supernatant was added to a 250 mL Erlenmeyer flask in 5 mL of 50% fresh medium solution of the composition shown in Table 2 (v / v). ) And incubated with shaking at 30 ° C. at 200 rpm for 5 days.

After culturing, the culture solution was recovered and the dry weight of the cells and microbial cellulose was measured in the same manner as in Example 2. As a result of the measurement, the dry weight of the cells and microbial cellulose in the medium not containing ethanol was 2.33 g / L and 1.30 g / L, respectively, 1.4 and 1.8 times lower than those in the medium containing ethanol, respectively. In the above results, the production of microbial cellulose was found to be higher in the medium containing ethanol.

Comparative Example 2: Mutant strain not producing microbial cellulose in a medium not containing ethanol (Cel ^-  mutant occurrence

In the same manner as in Example 4, the supernatant of the preculture solution was inoculated in 50% of the medium containing no ethanol at 5% (v / v) and incubated at a rotational speed of 200 rpm at 30 ° C. After diluting 10 to ⁵ times with saline, the solution was plated on a solid medium having the composition of Table 3 and incubated at 30 ° C. for 2 days. The results are shown in Table 7 below.

Incubation time (day) Total viable count (CFU / mL) Frequency of mutant strain (Cel ^- mutant / total cell) One 2.3 × 10 ⁶ 0.26 2 1.22 × 10 ⁸ 0.19 3 950 0.042 4 0 - 5 0 -

Table 7 and Table 4 show that when 1% (v / v) of ethanol is added to the medium, it is possible to reduce the incidence of cellulose (Cel ^- mutant) that does not produce cellulose during the culture.

Comparative Example 3: Production of Microbial Cellulose Using Continuous Batch Culture in Medium without Ethanol

The results are shown in Table 8 after the continuous batch culture in a medium not containing ethanol in the same manner as in Example 5.

Incubation time Dry weight of microbial cellulose (g / L) Number of batch cultures 1 time Episode 2 3rd time 4 times 5 times 1 day 1.26 0.55 0.18 2 days 1.26 0.66 0.49 3 days 1.32 1.40 1.32 0.63 0.28 5 days 1.46 0

As shown in Table 8, when continuous batch culture of the microorganisms cultured for three days in a medium containing no ethanol, it was found that the microbial cellulose production capacity is sharply reduced after three batches.

As described above, the present invention has an effect of providing a method for producing microbial cellulose, which has an improved production yield and an easy separation process. In addition, when the microbial cellulose is produced using the beer fermentation waste yeast liquid as a medium, it is possible to reduce the processing cost of industrial waste from the fermentation industry and to reduce the cost of cellulose production, thereby enabling the commercial production of microbial cellulose.

1 is a diagram showing the phylogenetic tree of the isolated strain.

Figure 2 is a diagram showing the form of microbial cellulose produced in a medium containing ethanol.

3 is a schematic diagram of a continuous batch culture process according to the present invention.

Figure 4 is a graph showing the production of microbial cellulose with culture time in beer fermentation waste yeast medium.

<110> PARK, Joong Kon <120> Method for Producing Bacterial Cellulose Using the Waste of Beer Fermentation broth <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1376 <212> DNA <213> Gluconacetobacter hansenii <400> 1 catgcaagtc gcacgaacct ttcggggtta gtggcggacg ggtgagtaac tcgtagggat 60 ctgtccatgg gtgggggata actttgggaa actgaagcta ataccgcatg acacctgagg 120 gtcaaaggcg cgagtcgcct gtggaggaac ctgcgttcga ttagctagtt ggtggggtaa 180 aggcctacca aggcgatgat cgatagctgg tctgagagga tgatcagcca cactgggact 240 ganacacggc ccagactcct acgggaggca gcagtgggga atattggaca atgggcgcaa 300 gcctgatcca gcaatgccgc gtgtgtgaag aaggttttcg gattgtaaag cactttcagc 360 ggggacgatg atgacggtcc ccgcagaaga agccccggct aacttcgtgc cagcagccgc 420 ggtaatacga agggggcaag cgttgctcgg aatgactggg cgtaaagggc gcgtaagcgg 480 ttgttacagt cagatgtgaa attcccgggc ttaacctggg ggctgcattt gatacgtgac 540 gactataatg tgagagaggg ttgtggaatt cccagtgtag aggtgaaatt cgtagatatt 600 gggaagaaca ccggtggcga aggcggcaac ctggctcatg actgacgctg aggcgcgaaa 660 gcgtggggag caaacaggat tagataccct ggtagtccac gctgtaaacg atgtgtgctg 720 gatgttggat ggcttggcca ttcagtgtcg tagttaacgc gataagcaca ccgcctgggg 780 agtacggccg caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 840 atgtggttta attcgaagca acgcgcagaa ccttaccagg acttgacatg cggaggctgt 900 gtccagagat gggcatttct cgcaagagac ctccagcaca ggtgctgcat ggctgtcgtc 960 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc gcctttagtt 1020 gccagcacgt ctgggtgggc actttaaagg aactgccggt gacaagccgg aggaaggtgg 1080 ggatgacgtc aagtcctcat ggcccttatg tcctgggcta cacacgtgct acaatggcgg 1140 tgacagtggg aagccaggca gcgatgccga gcggatntcc aaaagccgtc tcagttcgga 1200 ttgcactctg caactcgagt gcatgaaggt ggaatcgcta gtaatcgcgg atcagcatgc 1260 cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagttggttt 1320 gaccttaagc cggtgagcga accgcaagga cgcagccgac cacggtcggg tcagcg 1376

Claims (6)

Acetonitrile bakteo (Acetobacter) in, gluconic acetonitrile bakteo (Gluconacetobacter) genus, Agrobacterium (Agrobacterium), A separation tank emptying (Rhizobium) in and Pseudomonas (Pseudomonas) in the fermentation of microorganisms with selected cellulose-producing ability from the group consisting of beer waste Method for producing microbial cellulose, characterized in that cultured in yeast liquid. The method of claim 1, wherein the microorganism is Gluconacetobacter hansenii PJK (KCTC 10505BP). The method of claim 1 or 2, wherein the culture is a continuous batch culture. The method of claim 3, wherein the continuous batch culture comprises the following steps: (a) inoculating the culture supernatant with beer fermentation waste yeast after pre-culture of the microorganism having the ability to produce cellulose; (b) culturing the microorganisms in beer fermentation waste yeast to produce microbial cellulose in the form of tubers; (c) inoculating a portion of the culture supernatant with fresh beer fermented spent yeast solution; (d) producing microbial cellulose from the inoculated culture solution; And (e) repeating steps (c) and (d). Gluconacetobacter hansenii PJK (KCTC 10505BP) having cellulose- producing ability . A method for producing microbial cellulose, comprising culturing Gluconacetobacter hansenii PJK (KCTC 10505BP) according to claim 6 in a medium containing 0.5 to 15% (v / v) ethanol.