Start fermentation - (Oct/22/2014 )

Hello Everyone

I am again asking for a word of advice.

 

I am supposed to be doing fermentations soon. My problem is I never really had much experiences with these.

 

I need to run an easy fermentation like this.

 

Yeast DSM 1333

Yeast + Gram positive contaminant (Lactobacillus fermentum)

Yeast + Gram negative contaminant (E.coli)

 

I decided to use these conditions for 3 fermentations

 

Broth= Water 1L, yeast extract 10g/L, glucose 20g/L, peptone 20g/L

pH = 6

temperature 30C

suddenly my starter culture didnt even grow. Im using aeration now to see how it goes.

 

My doubt resides here I want to end with contaminants of 1.0x10^5 ufc/ml at least and final yeast of 1.0x10^8ufc/mL. how do I achieve that?

I know I have to use an hemacytometer to determine the amount of yeast initialy but what volume should i pour to start the fermentation?

and same thing for my bacteria... I have OD600=2 for both bacteria. what math do I do to know the volume I need to pour initialy as well and start fermentations?

 

Lets say I counted my yeast 1.2x10^5 ufc/mL. And if I start fermentation at 10^5 ufc/ml what volume should I pour? Is it by following the formula 

initial concentration x volume = final concentration (start fermentation) x volume of fermentation (250mL) would this be right?

 

but what is the formula to find out this wanted volume for the bacteria in OD=2 (can I use the hemacytometer to count bacteria? isnt that going to be really hard to do?)

 

 

Thank you for your time

 

 

Adriana

 

 

 

 

anyone has any experience in fermentations?

Adriana Reis on Tue Nov 4 09:45:26 2014 said:

anyone has any experience in fermentations?

 

Hi there.  I'm a professional brewer.  

 

First off, test your yeast.  Cheap way to do this is to make some wort and propagate 500 ml.  SG 1.040 is perfect.  You'll have to do the research on how to get this.  Boil some brewers dry malt extract or liquid malt extract.  A couple of teaspoons, but again, research this to see how much you'll need.  Pitch and ferment at 25°C.  Ironically, depending on how much you pitch, you'll have to wait up to 3 or 4 days for the population to get up to where you can see it.  

 

S. cerevisiae is essentially baker's yeast, so you can save some cash on doing some calculation trials using baker's yeast envelopes.  Freeze them.  

 

With this solution, you can do some trial dilutions for populations, using your hemocytometer.  Design a master spreadsheet for dilutions and the counting to calculate everything.  

 

Normal population curves for this type of thing is a 3-generation increase.  Typically, you'll find 1 of those 5 gram envelopes being used to pitch 20 litres of wort at 1.045 SG.  That is underpitching.  Double it and you'll get something reasonable.  Pitch rates can vary, and I've used a 400 ml yeast slurry to pitch 20 litres as well.  

 

pH should be in the 5.0 range.  pH will dip to 3.8-4.0 during fermentation.  Dissolved oxygen for proper pitch rates is 6-8 mg/l for proper cell membrane function.  Pitch rates are at 10^7 cells/l.  Be careful of warm fermentations as they can get violent and change your calculations post-fermentations due to krausen, which is a froth that takes out protein and yeast cells.  I would aim for 18-22°C optimally.  The lag phase happens post-pitch, so expect it.  24 hours you should see some buildup of cells and some fermentation action.  

 

It's best to get the viability of your yeast strain checked by propagating some in a regular media (wort) to begin with.  That is, if viability isn't what you're checking here.  I would suggest the same for your bacterial cultures as well.  

 

I'm not really sure of your application, so go ahead and ask away.  

 

HTH

 

Cheers

Sorry, some other notes.  

 

Bacteria can be counted, but you'll need the proper oil immersion oculars.  Obviously G+ will be easier to count.  You might need to use angle of inference of your light source to decipher where they are.  Be careful of not drying out your sample with the light source.  

 

Your spreadsheet will be your go-to for answers.  This is a numbers game based on hemocytometer calculations and dilutions.  It's a tough game.  Remember that bacterial cultures can replicate in 20 minutes in optimum conditions.  

Oh thank you! I started worked on it a long time ago. And I have more knowledge now. But still thanks!!!