How to tell if it's aerobic?

[Dotsie]

Hi all

 

I've done some work with an aerobic flask culture of E. coli, and so far so good. But my supervisor has asked me to prove that the culture is definitely aerobic before I write up. The volume is 10% of the flask volume, and has been shaking the whole time for a maximum of 24 hours, so it should be fine. But he said there's a simple test I can do, similar to a viologen-type test. And I can't find out what it is! It's proving to be very difficult to do an internet search, so any help would be appreciated.

 

Thanks in advance.

[CharonY]

Heh, I could think of half a dozen ones. Though the question is do you want to know whether it can survive oxygen, or whether it requires oxygen?

[Mr Skeptic]

I'm not much of an expert, but I'd say that if it can grow in Brewer's Anaerobic Agar, it's anaerobic.

[fredrik]

I'm no expert either but E coli are usually, like brewers yeast, facultative anaerobes, meaning that can do both respiratory growh and grow by fermentation. In brewers yeast, the same strain the homebrewer propagates mainly fermentative (the small amount of O2 supplied, are to build UFAs and ergosterol for healthy cellwalls, it's a common homebrewers myth that yeast respires during wort aeration, it doesn't becaues wort glucose level inhibits respiratoty pathways), is propagated commercially at the yeast companies aerobically.

 

Like CharonY already asked I think the question is what exactly that is to be proven. I might suspect that the question is to prove that the strain you have is not a respiratory-deficient mutant? And wether you have a pure strain at all? In normal brewers yeast for example, theres often a small % of the cell population that's respiratory-deficient. Some yeast propagation methods, unintentionally select for these, which usually give undesirable flavours.

 

I'm not very fluent in standard chemical tests used in modern labs since my experience is limited to what I can access at home, but usually biomass calculations gives info on the energy production and thus respiration/fermentation ratio. Without respiratory pathways, there is limit of possible biomass yield. With yeasts the respiratory deficient cells usually also look different in microscope.

 

There are tests wehre you can track the consumption of O2 in the culture, but from what I know of brewers yeast, the fact that o2 is consumed, does not mean it is used for respiratory energy production. THere are many places for o2 to go. So I don't thinks this alonw would prove anything either?

 

In beer fermentation, the wort is saturated with O2, and in very short time it's all gone, but many tests, including gene expression tests shows that while most O2 is consumed by yeast, rather than wort oxidations, none is used by respiratory pathways.. UFA and sterols synthesis are majors ones, but also pathways that disspiates excess o2 to reduce oxidative stress are in play. But all cellular processes are powered by fermentative pathways.

 

/Fredrik

[Dotsie]

Thanks for your replies. What I need to prove is that the E. coli, a known facultative anaerobe, is definitely growing aerobically, ie expressing only aerobic proteins and no anaerobic ones (which would invalidate my work so far). It's quite easy for a flask culture to become anaerobic if its too full or too old, and of course you can't tell by looking.

 

So the test needs to be done on the actual culture itself - subculture to any other medium would only prove what the bacterium is actually capable of, not what it's doing at the present.

 

So, any ideas? Keep 'em coming!

[CaptainPanic]

Just close off the fermenter, and see if growth continues?

 

If growth is purely aerobic, then there should be a (linear?) relation between growth and oxygen supply... right?

Disclaimer: I'm a chemical engineer, not biochemical.

[fredrik]

aerobically, ie expressing only aerobic proteins and no anaerobic ones

...

So the test needs to be done on the actual culture itself - subculture to any other medium would only prove what the bacterium is actually capable of, not what it's doing at the present.

 

I guess the resident expert may have better advice but what come to my mind is to look at the fermentative pathways in E coli and try to make sure none of the fermentation products are produced. Wether its' lactic acid, ethanol or anything else (I've never studied E Coli metabolism, just S.Cerevisae). If it does pure respiratory growth, no fermentative products should appear, right?

 

Other than that, some papers on yeast I've read does a modern gene expression analysis. You pull cells out of the culture and does an check what genes are expressed. But that seems like more high tech to me.

 

I'd look for production of compounds hat are only produced by the fermentative pathways, if you want to exlude that there's both fermentation and respiration going on at the same time.

 

/Fredrik

[CharonY]

Dotsie, thank you for the clarification. See, what you said makes a whole world of difference beacause you ask whether the cultures in question are currently performing aerobic respiration. Now the tricky part here is that a part of the culture could do it, whereas the others don't. This is especially likely in larger volumes. The thing is how sensitive the test needs to be. It depends on the the composition of your medium, though. I will assume that you used glucose as C-source, in this case you could simply add a pH indicator to monitor acidification due to fermentation. Depending on the overall media composition, though, even aerobic respiration might shift the pH somewhat, so for larger and older cultures this is a bit tricky. If you use a more sensitive method (as RT-PCR) you will generally find that in very old and thick cultures and very there is almost always a portion of the population either switching to anaerobic metabolism, or at least express mRNA generally associated with oxygen limitation. Even in absence of fermentation products.