Question
Asked 3rd Nov, 2015

Why is the pH increasing during fed-batch fermentation?

Hello everyone,
today I started a fed-batch fermentation with E. colis. Shortly after the induction with IPTG I recognized an increase in pH (as the acid pump had to work to keep the pH at 7). Unfortunately, I just can't figure out the reason why this is happening. Another group is doing the same and their pH stays constantly an 7.
Has it something to do with dying bacteria and release of basic compounds?
Thanks in advance :)

Most recent answer

11th Mar, 2017
Alireza Chackoshian Khorasani
Ferdowsi University Of Mashhad
During fermentation, pH usually decreases but it increases after a period. This is due to microorganisms consumed the nutrients and produced organic acids released into the medium, thus pH decreased. After the period, microorganisms face the lack of nutrients and begin to consume the organic acids as the nutrients sources, thus pH increased.
6 Recommendations

Popular Answers (1)

5th Nov, 2015
Christoph Wittmann
Universität des Saarlandes
Typically, if you have complex medium ingredients, they are composed of protein/peptides. LB, as example, is made from yeast extract and trypton. When cells use these nutrients, they cleave off ammonia from the contained amino acids, as they have a much greater demand for the carbon then for the nitrogen contained therein. Ammonia is released. In consequence, ammonia attracts a proton to form ammonium in aqueous solution - that is simple chemistry, which causes the pH increase.
13 Recommendations

All Answers (23)

3rd Nov, 2015
Christoph Wittmann
Universität des Saarlandes
How is your medium composition and which type of product do you have?
3rd Nov, 2015
Sven Be
Johannes Gutenberg-Universität Mainz
I used LB medium and added 2.2 g glucose /h. We expressed a protein (MetAP).
4th Nov, 2015
Zakaria Al-Qodah
Al-Balqa' Applied University
Usually, pH tends to  decrease in a fermentation becuase some of the products are acidic.  However, in this case, pH rises. This indicates that the cells were not able to utilize the carbon source or it may be at high concentration to inhibit and kill the E. coli. For this reason a lot of basic compound are produced from the decomposition of the cells.   
4th Nov, 2015
Alireza Chackoshian Khorasani
Ferdowsi University Of Mashhad
at the first, be sure about its pH sensor. in addition, in parallel, run a similar condition of fed-batch in the erlenmeyer flask-scale using shaker incubator.
4th Nov, 2015
Jai Ghosh
Smt. K. W. College Sangli 416416. Maharashtra. INDIA
Check whether the biomass production after each aliquot of  medium addition is as per you expectation. I presume that there is no contamination in the system. It is only showing a very significant lag which is slowing the growth. 
4th Nov, 2015
Jobrun Nandong
Curtin University Sarawak
Could be due to the malfunctioning pH sensor. What is the pH of the feed, above 7 or below 7? If the feed pH > 7, so that would cause the acid being added to regulate the bioreactor medium at pH 7, of course, assuming low microbial activity producing CO2 or/and other acidic products. If feed pH < 7, yet more acid was added to the bioreactor - well, this is unusual. Better redo the experiment, as quite often that an unusual event is due to human errors, or could be a slim case of something extraordinary, yet to be discovered!
5th Nov, 2015
Sofia Qaisar
Pakistan Council of Scientific & Industrial Research
same situation happened with bacterial batch type fermentation in lab. finally i put the culture  in refrigerator for a 20 days in 1% nutritive solution and this tip worked well. 
5th Nov, 2015
Christoph Wittmann
Universität des Saarlandes
Typically, if you have complex medium ingredients, they are composed of protein/peptides. LB, as example, is made from yeast extract and trypton. When cells use these nutrients, they cleave off ammonia from the contained amino acids, as they have a much greater demand for the carbon then for the nitrogen contained therein. Ammonia is released. In consequence, ammonia attracts a proton to form ammonium in aqueous solution - that is simple chemistry, which causes the pH increase.
13 Recommendations
5th Nov, 2015
Xuejia Liu
University of Waterloo
If possible you could always check your other fermentation productions instead of the protein by using a GC or HPLC, normally the pH increase was due to the production of ethanol. If the biomass decreased, then the pH shouldn't change.
5th Nov, 2015
Carlos Araújo Queiroz
Universidade NOVA de Lisboa
In case you have implemented a pH-stat control strategy, please note that a significant addition of isopropyl-β-D-thiogalactopyranoside (IPTG), which is acidic, may be able to decrease the pH of the fermenter broth, say, to around 6. In such circumstances, the pH-stat controlling system may eventually first add basic solution to increase the pH, and that may possibly cause a pH overshoot, particularly for a relatively slow stirring rate. If that occurs, the pH-stat system may then start to add acid solution. Eventually, pH may decrease to about 7, and if closely match the set-point, addition of acid solution may decrease and eventually stop. This being the case, it should be noticed that only if the addition of the acid solution continues steadily without addition of basic solution, while the pH remains nearly neutral, it can be safely concluded that basic compounds are predominantly generated, this leading to a steadily inherent tendency for the basicity of the broth to increase. 
6th Nov, 2015
Martin Gustavsson
KTH Royal Institute of Technology
I have to second the comment made by professor Wittmann. In a defined medium, like M9, you see a pH decrease due to a combination of acid production (mainly acetic acid) and consumption of ammonia as a nitrogen source, which removes a basic component from the medium. In a complex medium like LB, however, cells will utilise amino acids as a carbon source, leading to a net release of ammonia to the medium. This results in an increase in medium pH as long as amino acids are used as a carbon source.
8 Recommendations
6th Nov, 2015
Emmanuel Menya
Gulu University (GU)
In the initial period of fermentation, as large amounts of organic acids are produced by acid forming bacteria, the pH inside the digester can decrease to below 5. This inhibits or even stops the fermentation process.  However, as the digestion process continues, concentration of NH4 increases due to digestion of nitrogen, which can increase the pH value to above 8. In your case, you could think of lowering the pH by adding acid rich materials like foodwaste
1 Recommendation
9th Nov, 2015
Maizirwan Mel
International Islamic University Malaysia
Please check your bioreactor system, is it control system well functioned or not?. pH probe calibration from beginning, buffer system (acid and alkali) controller. Please check carefully.
10th Nov, 2015
Tapan Kumar Mazumder
YAEGAKI Bio-industry, Inc.
Please check your pH-stat strategy again, by using the data obtained from a batch fermentation by using the same medium and pH controlling agents. Depletion of carbon souce may lead to pH shoot up. Good luck
11th Nov, 2015
Seyed Ahmad Ataei
Shahid Bahonar University of Kerman
during the product , it may increase or decrease
12th Nov, 2015
Francesco Zimbardi
ENEA
which kind of fermentation/broth are you testing?
12th Nov, 2015
Aleksei Rozkov
YO Proteins AB
Another vote for pH increase due to use of amino acids in LB as a nitrogen source. pH was initially falling because of glucose fermentation into acetic acid and when it was exhausted pH went up. It makes sense. Also cell lysis after hard induction by IPTG is likely
1 Recommendation
9th Mar, 2017
Alok Malaviya
Christ University, Bangalore
I agree with Prof Wittman.
10th Mar, 2017
Tapan Kumar Mazumder
YAEGAKI Bio-industry, Inc.
I think Profs. Whiittman & Gustavsson explained clearly and nicely the mechanism of pH shift in relation tof media composition, during the course of fementtion. As you mentiond the other group doing the same is not experiencing the pH shift as yours. Please check if all the conditions were same as yours: the purpose of the fed-batch culture, the culture strategy, medium composition, inoculum size and its age, how long to continue the fed-batch culture,  etc. and Accordingly,  you better design your culture medium and a strategy to control pH f\to continue the culture as planned. Basic informations of the bacteria and culture medium medium should come from batch culture. Good luck.

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