Genetic study on locally isolated Gluconacetobacter xylinus and its ability in cellulose production

number: 
2903
English
Degree: 
Author: 
Noor Dheyaa Hameed
Supervisor: 
: Dr. Majed H. Al-Gelawi
: Dr. Hameed M. Jasim
year: 
2012

For the isolation of Gluconacetobacter xylinus, different food samples were collected from local markets in Baghdad governorate. These samples includes 66 vinegar samples (dates vinegar, date syrup vinegar and apple vinegar), and 54 rotting fruit samples (apple, peach and grape). From the overall of 120 samples a total of 105 isolates were obtained, seventy four of them were identified as Gluconacetobater spp according to their ability in acetic acid production. These isolates were further identified by subjecting them to morphological, microscopical characteristic and biochemical tests. Results showed that six of these isolates were belonged to Gluconacetobacter xylinus. Ability of these isolates on cellulose production was examined by culturing in (Hestrin-Schram (HS) medium) production medium and incubated at 30°C for one week. Results showed that all these six isolates of G. xylinus were cellulose producer with variable degrees. Among them, one isolate symbol G. xylinus N2 was the most efficient in cellulose production according to high productivity of cellulose (5.1 g/L) in its culture filtrate in comparison with cellulose productivity of the other isolates. Antibiotic susceptibility of G. xylinus N2 was examined against nine antibiotics. Results showed that this isolate was resistant to cephalexin, chloramphenicol, erythromycin, nalidixic acid and penicillin G, while it was sensitive to ampicillin, bacitracin, gentamycin and tetracycline. Plasmid profile of G. xylinus N2 was studied. Results showed that this isolate harbour only one plasmid and this plasmid is not responsible for cellulose production. This was examined throughout curing of plasmid DNA by using acridin orange as curing agent. The cured isolate was able to produce cellulose, but its ability was decreased to 1.82 g/L in comparison with the productivity of wild type (5.1 g/L), and this may be attributed to the regulatory role of the plasmid of G. xylinus N2. In order to enhance the ability of G. xylinus N2 for cellulose production, this isolate was subjected to random mutagenesis by using UV ray. After irradiation, one hundred colonies were selected randomly from the treatment, which gave approximately 90% killing and screened for their ability to produce cellulose. Results showed that only two enhanced producer mutants (G. xylinus N2_87 and G. xylinus N2_90) were obtained and the productivity of cellulose in their culture filtrate was 6.3 and 6.9 g/L (about 26% and 38%) respectively. Optimum conditions for cellulose production by the enhanced producer mutant G. xylinus N2_90 were studied. Results showed that the optimum conditions for cellulose production are growing this bacteria in production medium (HS medium) containing date syrup (2%) as a sole source for carbon and energy, yeast extract (2%) as a nitrogen source, in an initial pH 6.5 and incubation at 30°C for one week. Under these conditions, cellulose dry weight in culture filtrate of G. xylinus N2_90 was 8.5 g/L (about 23%).