M9 Media Broth turning green - (Feb/19/2014 )

Hi,

I am trying to isolate tungsten utilizing bacteria from soil sampled from a Tungsten mine. I did a serial dilution of the soil into LB first, streaked them and picked the colonies into M9 Minimal Media having varied conc of tungsten. I further incubated it for the purpose of taking the 0 hour, 24 hour and 48 hour readings. After the first 24 hour, the M9 media for one of the isolates turned green. And then after 72 hours, a lot the other broths turned green. Please let me know if this is a case of fungal contamination? 

What tungsten salt are you using and how do you envision it being "utilized"?  Tungsten oxide is typically yellow.  Have you observed any fungi?  Maybe you have a pyoverdin/pyocyanin producing bacterium such as P. aeruginosa.

Did you prepare any non-inoculated control flask? Check it. Did you add the W before or after autoclaving? Before or after adjusting pH?

If they are fungi are quite easy to distinguish, most of them form aggregates. Or just take a drop and look under the microscope.

In any case, I would first ask what do you mean by utilise tungsten... a first culturing in LB will select for heterotrophs and unless you get anything that can grow use CO2 as well as organic compounds as C-source, you shouldn't get anything as minimal media has no C-source... and the media vs mine soil have you considered the pH? you may be enriching for bugs that are not growing in soil conditions. If the mine produces scheelite the soil may be alkaline if the ore is skarn or if they mine wolframite it could be quite acidic due the presence of associated sulfides in the ore

Good points, El Crazy. Possible our colleague is seeing an overproduction of sideorphores mentioned as bug responds to the starvation conditions.  Many pseudomonads can grow in distilled water, you may not even find substantial growth.

Certainly even distilled water always have enough "crap" to allow the growth of some bugs, although it would be quite unusual to happen so fast. I mean if you don't specifically add a C-source, heterotrophs that can grow in such poor conditions are rather slow-growing. I even had fungi growing in my bottles of acidic water (just MQ pH 1.8 with sulfuric) but they took few months to even appear.

In any case I have no experience with the pseudomonads you mention and if you are not very careful cleaning the glassware, for some bugs is more than enough with some traces.

A photo of the cultures would be great, at least for curiosity :)

Pseudomonads (esp. B. cepacia) are the most troublesome contaminants of purified water systems.  Growth is fairly rapid - to 10E5 to 10E6 cfu/ml and typically forming biofilm.

http://www.ncbi.nlm.nih.gov/pubmed/811168

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC380831/

Thanks a lot for your replies! 

@Phil Geis. You asked me if I know whether the bacteria are utilizing tungsten or not. In fact, I don't know that yet. My primary goal is to establish this fact, for which I have isolated bacterial DNA, subjected them to 16S and have outsourced them for sequencing. The identity is yet to be established. Pseudomonas was actually my initial doubt. Actually a quite a few of the isolates did grow as a biofilm even on the broth media! Btw, thanks a lot for pointing me to the papers! 

@El Crazy Xabi. I added W after autoclaving and setting the pH. The mines contained WO3 mostly in the form of scheelite. LB is mostly alkaline. Do you think it is enough to first grow these bacteria on LB? If not, how can I prepare a media that has conditions similar to the soil samples of the mine?

Dear Sirs, actually I am just a master's student and I am performing these experiments as part my  six months dissertation project. I would appreciate it if you could guide me a little bit about the basics of bacterial isolation through the culturable approach. Now the thing about the so called "tungsten-utilizing bacteria" is that no specific biochemical tests exist for them. Could you suggest to me some method of establishing them as tungsten-utilizing bacteria? By tungsten-utilizing, I actually mean that certain enzymes utilize W as a co-factor. Even the ABC operon utilizes W.  Or my bacteria could simply be accumulating tungsten without utilizing them in their metabolism. Maybe my bacteria are anaerobes. I haven't checked that yet. 

My final aim would be to isolate such bacteria and test them for bioremedition. Thanks a lot again! I really value your opinion in these desperate times. 

Well, saying tungsten-utilising can be quite ambiguous. But if you are focusing in bioremediation I'll try to see any uptake/absorption or redox changes on the W.

The minimum requirement you want for those bugs is not only to be culturable but being able to develop in mining-like conditions. And that includes being able to withstand the W concentrations you will see there. Have you characterised the soil source? That's the first step. In (bio)geochemistry the pH is very important. I wouldn't be able to tell you with much detail because I don't know about your mine and the W cycling in that conditions, but I work with U in some related aspects, though I used mine waters as source of my bugs.

To get the best results I would start enriching in a selective medium with a similar pH, a given concentration of W (checking literature about MIC can help), and probably a soil extract (macerate or boil the soil source and use it as base liquid instead of MQ water or as trace element-source you have to find a protocol about it). The W can be added initially as a tungstate (Na2WO4 is soluble I think) and later as sterile scheelite powder if you can access to the mineral and if it's easier for you.

After enrichment, you will have to go for isolation on plates. At least keep the pH when you do it, specially if it is away from neutrality or you will start to culture the wrong bugs. Once you get them in pure cultures, you can proceed with the bioremediation test. I leave you some references I have though they are not for W they can give you an idea about what to do.

Good luck.

@El Crazy Xabi How do I check the uptake/absorption or redox changes on the W? Yes, I have characterized some of the soil source, did XRF analysis. I'm sending the remaining soil samples for either ICPMS or XRF soon. 

The pH of my soil samples are around neutral. ranges from 6.25 to 7.40. Let me know if it is OK to simply dissolve 1:5 ratio of soil to distilled water and take pH reading after shaking it for a bit. Since the pH of LB is also about 7, then is it OK to have cultured the bacteria into plain LB?

Sir, if it is not too much of a bother, then please tell me if the experimental design to check bioremediation of my bugs seems all right to you or not. I basically, plan on growing some plants under controlled conditions. Sodium Tungstate or Scheelite powder will be first added to the soil (with controls of course) and the plants will be tested for growth, biomass, chlorophyll content, free proline and carbohydrates. A peroxidase assay and an elemental analysis for tungsten spectrophotometrically will also be performed. Now, to check the bioremediation capabilities of my bugs, should I simply add the bacterial broth cultures to the W-laden soil in the plant pots and check for the same parameters mentioned above? This seems very crude, but could you suggest me anything better? 

Are you addressing tungsten uptake by plants and bacteria?  If so, how would you envision tungsten removal from the system - sedimentation with bacteria in sludge? Harvesting and disposal of plants?

Also, how do you see the sodium salt (vs. insoluble tungsten compound) as the primary environmental presence of tungsten?