Bob wrote:
> On Fri, 17 Dec 2010 12:14:05 -0500, Tom McCloud <tommccld from gmail.com>
> wrote:
>>> A few comments rather than answers:
>>>> Butanol is substantially soluble in water while oil not. Do you
>> know for a fact that your microbe survives in water containing a lot
>> of butanol? Perhaps demonstration of microbial growth in aqueous
>> media containing less butanol than what it takes to form a two-phase
>> system is in order. With only butanol as an energy source I would
>> speculate that growth will be slow.
>>>> What micronutrients are contained in your water?
>>>> Have you accidentally used the word 'hydrocarbon'? I would not
>> describe butanol as a hydrocarbon. I would describe butane, pentane
>> hexane and gasoline as hydrocarbons. T. McCloud
>> I agree with all that.
>> In addition, I would note that your growth conditions are not very
> good, and growth may be very slow. O2 supply is poor, since you are
> not mixing. Temperature is low. pH is low. (Perhaps you know what T
> and pH the bug needs.)
Adding my two pence in the Better-Late-Than-Never Department.
For a student, figuring out why the current system isn't working is
almost as, if not more, important than being spoon-fed one which
works...and learning how to correct failed experimental design (and
how to test for where the failing is) is an invaluable skill.
Those "kits" can be pretty cagey as to what bacteria are in their mix,
but I wouldn't be surprised find that it's not a single culture, but rather
a mixed bag comprised of pseudomonads, flavobacterium, arthrobacter
and/or azotobacter in order to maximise success under differing con-
ditions.
As may be, clearly the hydrocarbon is meant to be e- donor and, I sus-
pect, since this comes from a science supply place, oxygen is the e-
acceptor. Assuming that toxicity is not the issue (although I suspect
it probably is since a kit designed for consumption of food grade oil
is unlikely to contain critters able to utilise butanol) and assuming that
chemotaxis isn't a problem as all the above strains are reasonably mo-
tile, I would be inclined to wonder about e- acceptor limitation. The
original poster mentioned sufficient butanol to form a 0.25 inch "slick"...
without agitation, even diffusion is going to be inhibited over time...and
while there might be some more robust facultative members of the
consortium willing to accept less-than-stellar conditions, there's nothing
in the DI for them to work with.
I think my first step would be a down-and-dirty test for both toxicity
and limitation by bunging a bit of sodium nitrate (I'm not used to
working in Ball Jars, but perhaps 2-5 millimolar...maybe 40 mL of a
1M stock solution per jar) and see if any growth occurs under those
conditions.
Raising the temperature a bit wouldn't hurt either as most prefer to
be in the 25-30C range (say, 75-85 F)...at 6, the pH falls into the
"circumneutral" range, but it would probably be helpful to get closer
to 6.8-7.2 if possible (sodium nitrate may help a bit there).
If nothing happens after those variables are tweaked, then adding
agitation via shaker (or stir bar and plate) would be my next step
to completely rule out acceptor limitation. If there's still no growth
then the butanol is most likely the culprit and the donor needs to
be changed to something more amenable for growth like, perhaps,
a light motor oil. Or you could change your organism to some-
thing like Pseudomonas butanovora which has been demonstrated
to express 1-butanol dehydrogenases and is more likely to perform
in the manner desired.
Deirdre
