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Why the pH value decreased seriously in algal culture? - (Mar/03/2009 )

Hi Is there anyone who would be so kind as to give me some idea to explain this phenomenon?

I cultured several flasks of one green microalgae species in TAP medium under irradiance of 90 umol.m-2.s-1,
I added 18g/L glucose in the medium, and after 9days of cutlure, I found the aglal culture in bad growth condition though the biomass accumulated was much higher than the control culture without glucose, the color of the algae showed a tendency of turning white!! I collected the cells and tested the pH value of the culture medium, and it was below pH4.5!! I think most of the algal cells must have died! The pH value in the control culture without glucose was 8.0. I checked the culture under microscope and no bacteria was found.

I think the whole thing is very confusing :unsure:

The nitrogen source in TAP medium is NH4Cl, which should bring the pH down upon algal growth. So this could be a possible reason for the pH decrease in the above culture. But in another culture which also contained 18g/L glucose and under a much stronger irradiance of 800 umol.m-2.s-1. , the pH was 7.0 and with even more biomass accumulated!!! If NH4Cl is the criminal, then why the culture under stronger irradiance which should have taken in much more NH4Cl to produce biomass did not give a even lower pH??

Is it possible that the CO2 produced by respiration can cause a pH as acid as below 4.5?


The following is TAP recipe:


Tris - Acetate - Phosphate (TAP) Medium

(modified from Durnford Lab)


For One Liter of Medium:


2X Filnerís Beijernicks Solution 25ml

1M Potassium Phosphate 1ml

Trace mineral solution or 5ml

P IV solution 2X (see SVM) 3ml

Tris-Base 2.42g

Glacial Acetic Acid (17.4 mM acetate) 1ml

(pH should be 7.2)

Stock solutions:


2X Filnerís Beijernicks Solution (500 ml)

NH4Cl 8g

CaCl2 * 2H2O 1g

MgSO4 *7H2O 2g


Trace Mineral Solution (500 ml)

5 g disodium EDTA Ė dissolve in 400 ml water by heating and stirring

Neutralize to pH 6.5 with 5N NaoH

Add each of the following in order. Allow each to dissolve completely before adding the next.

0.5 g FeSO4*7H2O

2.2 g ZnSO4*7H2O

1.14 g H3BO3

0.51 g MnCl2*4H2O

0.016 g CuSO4*5H2O

0.073 g Na2MoO4*2H2O

0.016 g CoCl2*6H2O


1M Potassium Phosphate Stock (50 ml)

add: 20 ml 1M stock KH2PO4 (1M stock: 6.8g/50ml)

30 ml 1M stock K2HPO4 (1M stock: 8.7g/50ml)

pH 7.2 at RT

-biobunny-

your medium has 1mM phosphate and 20 mM tris (tris-acetate) buffers. buffering capacity is low and the resistance to change by tris is low because you are pushing the pH away from the pK.

since you add glucose, you are pushing respiration rather than photosynthesis. so you are releasing more co2 than you are using for photosynthesis.

this co2 dissolves in the medium and lowers the pH.

and, 4.5 is not as low as the pH can go in this system (unless all of your algae dies).

-mdfenko-

Thank you very much, mdfenko! :o

But I still feel confused. :lol:

I think what you mean is that the pH decrease was mainly due to the CO2 dissovled in the medium.
But I didn't get it why you said that "4.5 is not as low as the pH can go in this system"

It says that if the solution is open to the natural air which coantains 0.3% co2, the pH of saturated co2 solution is 5.6; and if the solution is in a sealed system, the pH of saturated co2 (1 atm) can go to about 4.0.
So only when the flasks was exclusively filled with CO2 which was continously produced by respiration, and the foil covered flask was not that efficient in air exchange, the medium pH went to as low as 4.0.

If that was true, after I uncovered the flask and the pressure of CO2 went back to the natural air level, why the pH in the medium did not rise to 5.6?


mdfenko on Mar 4 2009, 11:23 PM said:

your medium has 1mM phosphate and 20 mM tris (tris-acetate) buffers. buffering capacity is low and the resistance to change by tris is low because you are pushing the pH away from the pK.

since you add glucose, you are pushing respiration rather than photosynthesis. so you are releasing more co2 than you are using for photosynthesis.

this co2 dissolves in the medium and lowers the pH.

and, 4.5 is not as low as the pH can go in this system (unless all of your algae dies).

-biobunny-

biobunny on Mar 5 2009, 04:04 AM said:

It says that if the solution is open to the natural air which coantains 0.3% co2, the pH of saturated co2 solution is 5.6; and if the solution is in a sealed system, the pH of saturated co2 (1 atm) can go to about 4.0.
So only when the flasks was exclusively filled with CO2 which was continuously produced by respiration, and the foil covered flask was not that efficient in air exchange, the medium pH went to as low as 4.0.

If that was true, after I uncovered the flask and the pressure of CO2 went back to the natural air level, why the pH in the medium did not rise to 5.6?

because the co2 has interacted with the water to form carbonic acid which will take longer to outgas.

look at a carbonated beverage. it doesn't lose all of its carbonation immediately upon opening.

also, the co2 in the flask is at a higher percentage than the surrounding atmosphere.

keep in mind that the data you found was for solubility in water. you have other stuff present in the water that could also be interacting with the co2.

-mdfenko-

Thank you mdfenko :P

So you don't think it was the comsuption of NH4+ that caused the pH decrease?

Well, I will try to improve the aeration in the flask, maybe by trying sponge plug instead of foil cover frist.

However, my labmates culture algae heterotrophically in foil covered flask in dark, but they have not encoutered such a problem, I really wonder why they have not :)

-biobunny-

biobunny on Mar 5 2009, 07:04 PM said:

Thank you mdfenko :P

So you don't think it was the comsuption of NH4+ that caused the pH decrease?

Well, I will try to improve the aeration in the flask, maybe by trying sponge plug instead of foil cover frist.

However, my labmates culture algae heterotrophically in foil covered flask in dark, but they have not encoutered such a problem, I really wonder why they have not :huh:


just wondering, did your labmate also give his algae culture 18g/L of glucose? Was the aeration, culture size, flask size and type of flask (bevelled versus smooth) the same?

-perneseblue-

biobunny on Mar 5 2009, 10:04 PM said:

Thank you mdfenko :)

So you don't think it was the consumption of NH4+ that caused the pH decrease?

Well, I will try to improve the aeration in the flask, maybe by trying sponge plug instead of foil cover frist.

However, my labmates culture algae heterotrophically in foil covered flask in dark, but they have not encoutered such a problem, I really wonder why they have not :)

are your labmates using the exact same medium as you? they may have better buffering.

perneseblue, unless things have changed a lot in the past 32 years (the last time i cultured blue-greens (when they were still called algae)) they would be using ehrlenmeyer flasks.

-mdfenko-

Thanks to all who offered great suggestions!

However, I am still not sure about what caused the pH promble in my algal cutlure, may be it was just a result of multiple factors combinated situation--the foil cover unfavorable to areation, incubation shaker malfunction caused higher culture temperature and physically insufficient illumination....

And now with all culture conditions adjusted back to normal level, the problem seemed to be solved.

Thanks to all again, anyway!

-biobunny-