The present investigation demonstrates the effectiveness of ultraviolet (UV) radiation and ethyl methanesulfonate (EMS) in strain improvement for enhanced cellulose production by Gluconacetobacter xylinus NCIM 2526. The mutants were compared with wild type for cellulose production. UV mutants GHUV3, GHUV4, and GHUV5 of G. xylinus showed higher cellulose yield than the wild strain. The mutant GHUV4 gave cellulose yield of 3.92 g/l which was 30% more than the wild strain in standard medium. Chemical mutants GHEM4, GHEM6 and GHEM7 of G. xylinus showed higher cellulose yield than the parent strain (GHUV4). GHEM4 gave cellulose yield of 5.96 g/l which was 50% more than the parent strain (GHUV4) and 98% more than the wild strain (NCIM 2526). The results indicated that UV and EMS were effective mutagenic agents for strain improvement.Key words: Bacterial cellulose, Gluconacetobacter xylinus, ultraviolet mutagenesis, ethyl methanesulfonate treatment

Gupta, SG

Hungund, BS

English

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 African Journal of Biotechnology Vol. 9(32), pp. 5170-5172, 9 August, 2010     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals      Short Communication  Strain improvement of Gluconacetobacter xylinus NCIM 2526 for bacterial cellulose production  Basavaraj S. Hungund1* and S. G. Gupta2  1Department of Biotechnology, BVB College of Engineering. and Technology, Hubli, India. 2State Institute for Administrative Careers, Mumbai, India.  Accepted 24 May, 2010  The present investigation demonstrates the effectiveness of ultraviolet (UV) radiation and ethyl methanesulfonate (EMS) in strain improvement for enhanced cellulose production by Gluconacetobacter xylinus NCIM 2526. The mutants were compared with wild type for cellulose production. UV mutants GHUV3, GHUV4, and GHUV5 of G. xylinus showed higher cellulose yield than the wild strain. The mutant GHUV4 gave cellulose yield of 3.92 g/l which was 30% more than the wild strain in standard medium. Chemical mutants GHEM4, GHEM6 and GHEM7 of G. xylinus showed higher cellulose yield than the parent strain (GHUV4). GHEM4 gave cellulose yield of 5.96 g/l which was 50% more than the parent strain (GHUV4) and 98% more than the wild strain (NCIM 2526). The results indicated that UV and EMS were effective mutagenic agents for strain improvement.   Key words: Bacterial cellulose, Gluconacetobacter xylinus, ultraviolet mutagenesis, ethyl methanesulfonate treatment.     INTRODUCTION   Gluconacetobacter xylinus (formerly Acetobacter xylinum) has been known to produce pure cellulose for more than 100 years. The production of this bacterial cellulose (BC) is receiving great attention because of its unique properties and wide variety of applications. Due to its high tensile strength and water-holding capacity, BC has been used as raw material for producing high-fidelity acoustic speakers, high quality paper and diet and dessert foods (Ross et al., 1991; Iguchi et al., 2000). Moreover, BC could be used as artificial skin (Joris and Vandamme, 1993) and as a promising material for a potential scaffold for tissue engineering (Svensson et al., 2005). One of the BC application problems in industry is its low productivity. One approach to combat BC productivity is strain improvement of the producer organism. As yet, no total BC  synthesis   operons   have  been  cloned  or  no  cells    *Corresponding author. E-mail: bshungund@bvb.edu. Tel: +91-9449169349. Fax: +91-836-2374985.    Abbreviations: UV, Ultraviolet; EMS, ethyl methanesulfonate; BC, bacterial cellulose; HS, Hestrin-Schramm; NTG, N-methyl-N/-nitro-N-nitrosoguanidine. transformed with the BC synthesis gene (Romling, 2002). Ishikawa et al. (1995) obtained mutants of A. xylinum BPR 2001 resistant to sulfaguanidine by NTG (N-methyl-N/- nitro-N-nitrosoguanidine) treatment. They reported increased cell growth and cellulose production. The aim of the present investigation is to increase cellulose pro-ductivity of G. xylinus NCIM 2526 by ultraviolet (UV) radiation and ethyl methanesulfonate (EMS) treatment.    MATERIALS AND METHODS  Microorganism and growth media   The organism used was G. xylinus NCIM 2526 obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, India. The stock culture was maintained on Hestrin-Schramm (HS) agar slants, transferred monthly and stored at a temperature of 2 - 8°C in the refrigerator. The culture medium used was the standard Hestrin-Schramm (HS) medium (Hestrin and Schramm, 1954) which contains (w/v) 2.0% D-glucose, 0.5% peptone, 0.5% yeast extract, 0.27% disodium phosphate and 0.115% citrate. The cultures were incubated at 30°C in 100 ml quantity of the medium for 14 days. The pellicle formed after 14 days of incubation was removed carefully, boiled in 2.0% NaOH solution for 30 min and thoroughly washed with distilled water. Drying of the pellicle was carried out at 65°C in an oven for  6 h.  The  dry  weight  of  the cellulose was calculated.       Table 1. UV mutants and their cellulose yield.  S/N Description BC yield (g/l) 1 GHUV1 NIL 2 GHUV2 2.21 3 GHUV3 3.45 4 GHUV4 3.92 5 GHUV5 3.61 6 GHUV6 2.01 7 GHUV7 1.25 8 GHUV8 2.98 9 GHUV9 NIL 10 GHUV10 1.25 11 GHUV11 2.98 12 GHUV12 2.54 13 GHUV13 2.12 14 GHUV14 2.84 15 GHUV15 1.23 16 G. xylinus (wild strain) 3.04  Organisms were cultured for 14 days in 250 ml Erlenmeyer flasks containing 100 ml of standard HS medium.    Physical mutagenesis and mutant selection   Organism was grown in 50 ml of HS broth at 30°C for 24 h. Such transfers were made thrice to get good growth within 24 h. 20 ml of 24 h old culture broth was harvested by centrifugation at 5000 rpm for 10 min. The supernatant was discarded and the pellet was re-suspended in 10 ml of sterile phosphate buffer pH 7. The total viable count of the sample was found to be 107/ml. The suspension was further diluted to have viable count of 105/ml. 10 ml of the culture suspension (viable count 105/ml) was taken into a Petri plate of 100 mm diameter. The sample was irradiated with a germicidal lamp (UV light source) from a distance of 20 cm. Samples were withdrawn after 10, 20, 30, 45 and 60 s. These irradiated samples were serially diluted in phosphate buffer. The survivors were deter-mined by spreading 0.1 ml of the sample on screening medium (HS agar with 0.02% fluorescent brighter). Fluorescent brightener is a fluorescent dye that avidly binds to , D-glucans and cellulose producing colony fluoresces when observed under UV-light. A total of 15 colonies (designated as GHUV1 to GHUV15) were selected from the plates and tested for cellulose production ability. The results were compared with that of the wild strain.    Chemical mutagenesis and mutant selection  The best UV mutant (GHUV4) was used for chemical mutagenesis. The culture suspension was prepared in the same manner as described earlier. To 5 ml of cell suspension with viability of 105/ml, 5 ml of sterile solution of EMS (200 µgml-1) was added. The reaction was allowed to proceed. Samples were withdrawn from the reaction mixture at intervals of 15, 30, 60, 90, 120 and 150 min. Immediately, the samples were resuspended in sterile buffer pH 7. The suspended sample was again centrifuged at 5000 rpm for 10 min. The supernatant was discarded. Cells were washed thrice with sterile distilled water to remove traces of EMS. The samples were serially diluted in the same buffer and plated on  screening  medium  Hungund and Gupta   5171    as mentioned earlier. A total of 15 colonies (designated as GHEM1 to GHEM15) were selected from the plates and similarly tested for cellulose production ability.   RESULTS AND DISCUSSION   Strain improvement can generally be described as the use of any scientific techniques that allow the isolation of cultures exhibiting a desired phenotype. The technology has been utilized for more than 50 years in conjugation with modern submerged culture fermentations (Victor and Graham, 1999). There are only few reports of mutating A. xylinum for increased cellulose production. Ishikawa et al. (1995) subjected the cells of A. xylinum subsp. sucro-fermentans BPR 2001 to chemical mutagenesis by NTG (N-methyl-N/-nitro-N-nitrosoguanidine). The mutant BPR 3001E gave 40% higher cellulose than parent G. xylinum. They found that cellulose production was associated with growth.  We tried mutagenesis of the organism both by physical and chemical mutagenic agents. Bapiraju et al. (2004) reported mutation induced enhanced lipase production from Rhizopus sp. using UV radiation and NTG. Kadam and associates (Kadam et al., 2006) successfully employed UV mutagenesis to improve strain of Lactobacillus delbruekii for lactic acid production. EMS is a well-known mutagenic agent, whose mode of action is attributed to alkylation at nitrogen position 7 of guanosine of the DNA molecule, leading to transversion or transition type of mutations (Freese, 1961). Successful use of EMS in induced mutations has been reported for many bacterial strains (Shanthamma et al., 1972; Haq et al., 2009). The colonies of G. xylinus are typically small and convex in appearance, eventually developing uneven edges where the bacterial cells break through the cellulose. Mutant selection was based on growth rate, colony morphology and fluorescence on screening medium.  Wild strain of G. xylinus was subjected to UV muta-genesis. A total of 15 mutants were selected and screened for their cellulose production abilities (Table 1). Three UV mutants GHUV3, GHUV4 and GHUV5 produced 3.45, 3.92 and 3.61 g/l cellulose, respectively. The mutant GHUV4 gave maximum cellulose production of 3.92 g/l which was 30% more than the wild strain in standard HS medium. Furthermore, the best UV mutant GHUV4 was subjected to chemical mutagenesis. The mutants GHEM4, GHEM6 and GHEM7 produced 5.96, 5.62 and 4.89 g/l cellulose, respectively (Table 2). The mutant GHEM4 gave cellulose yield of 5.96 g/l which was 50% more than the parent strain (GHUV4) and 98% more than that of wild strain (NCIM 2526). Haq et al. (2009) reported strain improvement of Bacillus licheniformis for alpha amylase production using EMS. This is one of the rare reports of mutagenesis for increased cellulose production using UV radiation and EMS. Effectiveness of UV radiation (physical mutagen) and EMS (chemical mutagen) in strain improve-ment for enhanced cellulose production has been demon-  5172      Afr. J. Biotechnol.    Table 2. EMS mutants and their cellulose yield.  S/N Description BC yield (g/l) 1 GHEM1 3.56 2 GHEM2 3.54 3 GHEM3 2.54 4 GHEM4 5.96 5 GHEM5 NIL 6 GHEM6 5.62 7 GHEM7 4.89 8 GHEM8 2.54 9 GHEM9 1.28 10 GHEM10 1.85 11 GHEM11 3.79 12 GHEM12 2.78 13 GHEM13 2.55 14 GHEM14 1.98 15 GHEM15 3.01 16 GHUV4 (parent strain) 3.90  Organisms were cultured for 14 days in 250 ml Erlenmeyer flasks containing 100 ml of standard HS medium.     strated in this investigation.    REFERENCES  Bapiraju K, Sujatha P, Ellaiah P and Ramana T (2004). Mutation induced enhanced biosynthesis of lipase. Afr. J. Biotechnol. 3: 618-621.  Freese EB (1961) Transitions and transversions induced by depurinating agents. Proc. Natl. Acad. Sci. USA, 47: 540-545. Haq IU, Ali S, Saleem A, Javed MM (2009). Mutagenesis of Bacillus licheniformis through ethyl methanesulfonate for alpha amylase production. Pak. J. Bot. 41: 1489-1498.  Hestrin S, Schramm M (1954). Synthesis of cellulose by Acetobacter xylinum. II. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose. Biochem. J. 58: 345-352.                               Iguchi M, Yamanaka S, Budhiono A (2000). Bacterial cellulose- a masterpiece of nature’s art. J. Mater. Sci. 35: 261-270.  Ishikawa A, Masuoka M, Tsuchida T, and Yoshinaga F (1995). Increase in cellulose production by sulfa guanidine-resistant mutants derived from Acetobacter xylinum subsp. sucrofermentans. Biosci. Biotechnol. Biochem. 59: 2259-2262.  Joris K, Vandamme EJ (1993). Novel production and applications of bacterial cellulose. Eur. Microbiol. 1: 27-29.   Kadam S, Patil SS, Bastawde KB, Khire JM, Gokhale DV (2006). Strain Improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid production. Proc. Biochem. 41: 120-126.   Proc. Natl. Acad. Sci. U.S. 47: 540-545. Romling U (2002). Molecular Biology of cellulose production in bacteria. Res. Microbiol. 153: 205-212. Ross P, Mayer R, Benzimann M (1991). Cellulose biosynthesis and functions in bacteria. Res. Microbiol. 153: 205-212.  Shanthamma MS, Joseph R, Rao TNR (1972). Quantitative Assessment of the Mutagenic Effect of Ethyl Methanesulfonate in Micrococcus glutamicus. Folia Microbiol. 17: 81-87.  Svensson A., Nicklasson E, Harrah T, Panilaitis B (2005). Bacterial cellulose as a scaffold for tissue engineering of cartilage. Biomat. 26: 419-431. Victor AV, Graham B (1999). Strain Improvement by Nonrecombinant Methods. In Demain and Devis (eds) Manual of Industrial Microbiology and Biotechnology. ASM Press, Washington, D.C. pp. 103-113.    

Strain improvement of Gluconacetobacter xylinus NCIM 2526 for bacterial cellulose production

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Strain improvement of Gluconacetobacter xylinus NCIM 2526 for bacterial cellulose production

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