Increased Production of Microbial Cellulose by Ultraviolet Irradiated Acetobacter xylinum

Antonio B. Bustamante Jr. Lovely I. Flores Rex Salvador T. Tenazas Aiko F. Valeroso Jervene C. Venturina Bachelor of Science in Biology, Major in Biotechnology
1 5 MATERIALS AND METHODS Acetobacter xylinum Stock Culture Inoculation on HS Agar Medium Mutagenesis (UV radiation) Subculturing Fermentation in static condition Product Recovery and Purification Thickness and Weight Measurement

ABSTRACT A study was conducted to increase the production of bacterial cellulose by genetic improvement of Acetobacter xylinum using ultraviolet radiation as mutagen. Methodology for cellulose production includes obtaining stock culture of the microorganism and maintaining it in Hestrin-Schramm(HS) media; inoculation of the microorganism on 4 Petri dishes and incubation for 3-4 days at 25-30C prior to UV irradiation; subjecting the 3 Petri dishes to UV irradiation with different exposure times(15s,30s,45s), 20 cm from the lamp; subculturing of the UV irradiated microorganisms to test tubes and transfer to ketchup bottles with HS broth of 100 ml for cellulose production, divided into glucose and sucrose as their carbon source; product recovery and purification using NaOH. The increase in production was measured by using 4 criteria: thickness in mm after incubation; wet weight measurement in grams of the pellicle after purification; dry weight measurement in grams after oven-drying; and the yield of biosynthetic process which is the ratio of carbon input-output in the process using the formula Y = C / G * 100%. Results were almost parallel in all criteria. The bacterial cellulose in 45s is relatively higher and almost all UV treatments were higher than the control. However, there is no significant difference between the UV treatments and the control with regards to both graphical and statistical analysis. Between the two carbon sources, glucose is evidently better than sucrose based on both graphical and statistical analysis.

RESULTS AND DISCUSSION Two-way ANOVA of Thickness Measurement. = Critical Value Tabular Value Interpretation 0.05
0.223 15.246 9.28 10.1 No significant difference Significant difference

UV Treatment Carbon Source

Critical Value Tabular Value Interpretation Two-way ANOVA of Wet Weight Measurement. UV Treatment 0.801 9.28 No significant difference = 0.05 Carbon Source 240.898 10.1 Significant difference

Critical Value Tabular Value Interpretation Two-way ANOVA of Dry Weight Measurement. UV Treatment 6.006 9.28 No significant difference Carbon Source 149.609 10.1 Significant difference 0.05

UV Treatment Carbon Source

Two-way ANOVA of Biosynthetic Process. = 0.05

Critical Value 6.080 148.670

Tabular Value 9.28 10.1

Interpretation No significant difference Significant difference

Data analysis and Statistical Treatment

RESULTS AND DISCUSSION

UV treatments are relatively higher than control. 45 s strain is the best strain relatively Glucose is better than sucrose almost by half. Statistical treatment was done using 2-way Analysis of variance. Statistical results agree with graphical/tabular results: There is no significant difference between the UV treatments (15s,30s,45s, control) There is significant difference between the glucose and sucrose as carbon sources

2 INTRODUCTION Bacterial cellulose, produced by Acetobacter xylinum displays unique properties including high mechanical strength, high water absorption capacity, high crystallinity, and an ultra-fine fibre network structure. It is expected to be an extensive biochemical material for the industry with the variety and range of its applications. Thus, methods on how to improve its production have been extensively made for the past decade. One of the best way to enhance this process is by improving the microorganism itself using strain improvement which is the method for this study. Strain improvement was done in the present study by inducing mutagenenesis on the Acetobacter xylinum via UV radiation to increase its production of the highlyvalued bacterial cellulose. 3

Mean Thickness Measurement of the Pellicles

OBJECTIVES The study aims to increase the production of bacterial cellulose by genetic improvement of Acetobacter xylinum using ultraviolet radiation as mutagen. Specifically, it sought to determine the following: The amount of microbial cellulose in terms of thickness, wet weight and the dry weight measurement produced by the wild type and mutant strains. The comparison of using different carbon sources (glucose and sucrose). The yield of biosynthesis process by both wild type and mutant strains. The microscopic characteristics of both wild type and mutant strains. Significant difference between the UV treatments (exposure time) and between the two carbon sources by statistical treatment SIGNIFINACE This study will be a significant endeavor to increase the production of microbial cellulose by UV irradiated Acetobacter xylinum. Since bacterial cellulose has properties like high purity, high degree of crystallinity, high density, good shape retention, high water binding capacity, and higher surface area as compared to the native cellulose, it can be used in various areas including textile industry, paper, food, pharmaceutical, waste treatment, broadcasting, mining and refinery. Proving the effect of UV radiation on bacterial cellulose production of Acetobacter xylinum in this study can give more drive for industries to enhance their cellulose production using the said method

Mean Wet Weight Measurement of the Pellicles

Acetobacter xylinum (a) after 15 s exposure to UV (x100); (b) after 30s exposure (x100);(c) after 45s exposure (x100); wildtype (x100).

Mean Dry Weight Measurement of the Pellicles 8

Observation using light microscope shows that those treated with UV exposed for 45 seconds is of larger size than the wild type strain of Acetobacter xylinum and of the other subjected to UV with different exposure times.

Mean Yield of the Biosynthetic Process

CONCLUSION In terms of microscopic structure, the cells of mutant strain labelled as 45s appears to be relatively larger in size as compared to other mutant strain and wild type. Glucose is relatively a better carbon source than sucrose which is evident in all parameters used. It is supported by the statistical treatment showing significant difference between the variables There is an increase in the production of cellulose, however it is not significantly different when statistical means are concerned. RECOMMENDATIONS Different distance from UV lamp and exposure time More precise measuring devices Mutant screening and selection Other types of radiation like Gamma radiation

5/29/12

The yield of the biosynthetic process (Y) is calculated using the formula Y = C / G * 100, where C is the weight of dry film in grams and G is the weight of carbon source in substrate (2g/100ml)