pH: the weak are really strong

I sometimes get comments and usually these are of a nature that either require no response or a simple response. However, the other day I received a question/comment (quomment?) that I have decided to respond to by way of a post (which was started many months ago and completed today). In part, because this quomment allows me to expand on the issue of pH and biological systems.

First the comment, Hi, you mentioned that pH range for growth is 2-10. The growth of my Candida strain stops at about pH 4 - 5. I am not sure why this is the case, the media has a high content of glucose, though. Any suggestions? Does optimal pH for growth depend on the media? What is the best media for growth? Thanks so much for your help! posted by anonymous

What I think we are dealing with here is a "weak acid" versus "strong acid" issue. This came up in your basic chemistry classes, but if you are like me, you probably forgot about it as soon as the test came and went. So I apologize if this brings back painful memories, it actually turns out to be important.

Most organisms, including us live at or near pH 7. However, many organisms can survive and even grow at acidic pH, let's say pH < 6 and others like C. albicans can grow at pH 2! Now I can grow C. albicans at pH 2, but our commenter has trouble growing it at pH 4. This is only a difference in 2 pH units, but this is in fact a 100-fold difference. That's analogous to deciding between buying a $1000 used 1994 Ford Escort or a $100,000 BMW 7 series.







So, why would we be seeing a growth difference? I can think of 3 explanations: 1. Our strains are different, 2. Our growth media are different, 3. There's a technical issue that is the cause of this difference. I suspect that the difference is due to this 3rd issue. Now let's assume we are using the same strain and the same medium. We first need to adjust the pH of our medium and this is done by adding either acid or base depending on the starting and desired pH of the medium. Not surprisingly, since we are making media at pH 2 and pH 4, we will be adding acid. Ok, which acid? does it matter? and if so, why?

We routinely use hydrochloric acid (HCl) to make growth medium more acidic. However, another common acid used is acetic acid (CH3COOH, from here on out AcH). The differences are numerous, but 1M acetic acid has a pH of 2.4, whereas 1M HCl has a pH of 0. So both can be used to get growth medium to pH 4, but only HCl can be used to get to pH 2. These two chemicals also differ in their pKa (acid dissociation constant): AcH is 4.8 and HCl is -8.0 (remember that p in pKa is the mathematical symbol for -log, so the differences in the pKa's of AcH and HCl is ~10,000,000,000,000 (yep, that's 10 trillion!)). Now for those of you who do not want the gory chemical details pKa tells you how much acid is dissociated in an aqueous solution, like inside a cell or in a growth medium (see it will come back around). A pKa < -2 means that virtually all the acid is dissociated whereas higher pKas means that some of the acid is not disassociated and the higher the number the less that is dissociated. What does that mean? Well in general acids are acids because they yield H+ (pH is the measure of H+ in solution and acidic solutions have more H+s than basic solutions). So something like HCl in an aqueous solution does not exist as HCl but as dissociated H+ and Cl-. However, AcH in an aqueous solution exists as a mix of AcH, Ac-, and H+. HCl dissociates so readily that its pKa has only been determined theoretically! So regardless of pH, HCl is found as H+ and Cl-. However, depending on the pH, AcH can either be more or less dissociated. At acidic pHs, where there is an excess of H+ already, any AcH that dissociates to Ac- immediately finds another H+ to interact with reforming AcH. Conversely, at neutral or alkaline pHs, there are fewer H+s so when AcH dissociates to Ac- and H+, it stays that way. This then makes the solution more acidic. Ok, thats what you need to know: under the conditions we are working with HCl is always dissociated and AcH is a mixture, which depends on the pH.

One more thing you need to know. The plasma membrane of the cell does not allow charged molecules to cross into or out of the cell. A charged molecule is something with a + or -, like H+, Ac-, or Cl-. So, the plasma membrane keeps charged molecules in the environment out of the cell. However, small uncharged molecules, like AcH, can readily cross the membrane and get into the cell. See where this is going?

So lets go back to our standard growth medium, which we will assume has a neutral pH. If we add some AcH or HCl, the acid immediately dissociates to H+ and Ac- or Cl-, this lowers the pH a little, and if our organism can grow at that pH everything is cool. Let's add even more AcH or HCl to our growth medium to get the pH to 4. All the HCl is dissociated to H+ and Cl-, but these molecules cannot enter the cell, so as long as an organism is viable at pH 4, it should grow (as C. albicans does). However, not all of the AcH is dissociated to Ac- and H+. The remaining undissociated AcH, which is not charged, can cross the plasma membrane to enter the cell. The cytoplasm of the cell has a pH of ~7, so now the AcH dissociates to Ac- and H+! This acidifies the cytoplasm and kills the cell. Since dead C. albicans don't grow so well, no growth occurs. But realize that this is not due to an inability to grow at pH 4 in general (C. albicans grows in pH 4, and pH 2, medium using HCl), but an effect of weak acid stress.

I would highlight that this is not a trivial issue in microbiological labs. This is a common and historically important method of food preservation. Vinegar, which is mostly AcH, has long been used to preserve foods, like