I am doing sub-culture of my bacteria (WT) every 16 h in 5 ml LB broth with antibiotic for 30 days. For whole genome sequencing, I should change the LB broth sub-culture 5 ml to 100 ml to collect pellet from 10th day, 20th day and 30th day. I also carried spread plate technique (5 days once) for checking the morphology of bacteria. I did not find any variant (mutant) upto 9 days on LB plates. When I changed my culture volume 5 ml to 100 ml, I found WT bacteria (80%) as well as variant (20%). After 15 days, I found WT bacteria (20%) and variant (80%). After 20 days, I found complete variant on LB plates. Can anyone suggest the reason (nutrient starvation, LB volume, oxygen depletion) please? 

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