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help with saccharomyces glucose batch fermentation setup


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#1 tnad

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Posted 02 February 2009 - 01:06 PM

Hi, we've been trying to set up a batch fermentation of glucose using saccharomyces cerevisiae and are having some problems with cell growth. The way we do it is to just grow the cells in tubes with caps that allow some air and leave on a shaker at 30 C. We consider this a microaerobic condition since it's not completely sealed. We used 5% glucose. It appears that cells stop growing after 24 hrs and reach an OD600 of about 5. We are concerned that this setup is affecting the growth somehow. Has anyone done this type of experiments to get a strain that ferments glucose to ethanol well? If so, can you help us with the setup or post links that help? Any comments are welcome too. Thank you.

#2 GeorgeWolff

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Posted 02 February 2009 - 04:41 PM

Sorry - it sounds pretty casual. 5% glucose and what? What you may be doing ot exhausting the oxygen - forcing the yeast into anaerobiasis and growth under those conditions can't persist without supplementation of fatty acids. Try adding some tween 80.

#3 Julio-Claudian

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Posted 03 February 2009 - 06:45 AM

OD600nm of 5 (it's really dense).. Hmm... You measured the undiluted sample? Anyway, the yeasts could theoretically get "drunk" (something like end product inhibition) so perhaps growth reached a plateau.

See what you can come up with GeorgeWolff's suggestion. Good luck!

#4 tnad

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Posted 03 February 2009 - 10:33 AM

Sorry - it sounds pretty casual. 5% glucose and what? What you may be doing ot exhausting the oxygen - forcing the yeast into anaerobiasis and growth under those conditions can't persist without supplementation of fatty acids. Try adding some tween 80.


oh sorry, I should've had more information. It has 5% glucose, Difco™ yeast nitrogen base (without amino acids; with essential vitamins and inorganic salts) and amino acid mix (dropout mix). This experiment is basically to test our best engineered ethanol producing strain before we start with continous growth in a fermentor. We dilute to measure OD if it ges over 2. We also tried to buffer and start at a pH of 6.5, 6 and 5.5 but it didn't make much difference. The cells get to OD 1.4 in 12 hrs, 4.4 in 24 hrs, 4.4 to 5 in 48 hrs and remain about the same at 56 hrs.

Edited by tnad, 03 February 2009 - 10:41 AM.


#5 GeorgeWolff

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Posted 04 February 2009 - 04:18 PM

Saccharomyces cerevisiae can't grow indefinitely under anaerobic conditions without specific supplementation. Their mitochondria degenerate and absent their function fatty acid desaturases are not produced. Unsaturated fatty acids perform multiple functions including maintenance of membrane integrity and fluidity. Supplementation with unsaturated fatty acids (such as in Tween 80) replaces this loss. Even with supplementation, they grow slowly - on plates the smaller colonies are referred to as petite and you can induce petite mutants by screwing up mitchondrial function (e,g, with ethidium bromide).




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