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M9 Media Broth turning green

M9 Media Green Colour New Isolates Tugsten utilizing bacter

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#1 p.phadtare

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Posted 19 February 2014 - 12:40 AM

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? 



#2 Phil Geis

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Posted 19 February 2014 - 04:22 AM

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.


Edited by Phil Geis, 19 February 2014 - 08:36 AM.


#3 El Crazy Xabi

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Posted 19 February 2014 - 03:28 PM

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



#4 Phil Geis

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Posted 20 February 2014 - 07:07 AM

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.



#5 El Crazy Xabi

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Posted 20 February 2014 - 02:59 PM

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 :)



#6 Phil Geis

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Posted 20 February 2014 - 07:09 PM

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....v/pubmed/811168

http://www.ncbi.nlm....cles/PMC380831/



#7 p.phadtare

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Posted 23 February 2014 - 08:13 AM

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. 



#8 El Crazy Xabi

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Posted 23 February 2014 - 06:27 PM

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.

 

 

Fomina M, Charnock JM, Hillier S, et al. (2007) Fungal transformations of uranium oxides. Environ Microbiol 9:1696–1710. doi: 10.1111/j.1462-2920.2007.01288.x
Fomina M, Charnock JM, Hillier S, et al. (2008) Role of fungi in the biogeochemical fate of depleted uranium. Current Biology 18:R375–R377. doi: 10.1016/j.cub.2008.03.011
Fomina MA, Alexander IJ, Colpaert JV, Gadd GM (2005) Solubilization of toxic metal minerals and metal tolerance of mycorrhizal fungi. Soil Biology and Biochemistry 37:851–866. doi: 10.1016/j.soilbio.2004.10.013


#9 p.phadtare

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Posted 24 February 2014 - 08:30 AM

@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? 


Edited by p.phadtare, 24 February 2014 - 08:33 AM.


#10 Phil Geis

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Posted 24 February 2014 - 02:37 PM

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? 



#11 El Crazy Xabi

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Posted 24 February 2014 - 04:54 PM

There are quite of few options to evaluate the possibility of using the bugs you isolate for bioremediation. Microbes interact with geochemical cycles in so many different ways that it is hard to say what would be the best option for your project.

 

I cited the redox changes because some elements are quite prone to suffer redox transformations by organisms even if they are not involved in respiration. But looking to some Eh-pH diagrams for W, it is unlikely under neutral oxic conditions, but it will be different in anoxic sediments. This will help http://link.springer...1007/BF02650012

 

With a pH around 7 the question is if the W is really a problem. Is it movable? Can affect to xxx? Many remediation strategies are based on the transformation of some compounds or elements in less movable/soluble forms.

 

LB culturing as primary isolation method will select for some heterotrophic microbes disregarding if they are capable of withstand W or not. (by the way, culturing temp?) So, it is likely that you will have many colonies that won't be useful for the following tests.... unless you want to use the biomass as a biosorbent system to bind W and you don't care about the viability of the bugs in the soil. Although this approach is more common for treatment of effluents and liquid wastes. e.g. Omar, N. B., Merroun, M. L., González-Muñoz, M. T. & Arias, J. M. (1996). Brewery yeast as a biosorbent for uranium. Journal of Applied Microbiology 81, 283–287.

 

The measurement of the pH of a soil it is not something universally standardised, just be sure that all the measurements are performed in the same way.

 

plants too? O_o



#12 p.phadtare

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Posted 26 February 2014 - 09:34 AM

@Phil Geis I am primarily addressing tungsten uptake by bacteria in soil. In the same soil I decided to plant some species (of Brassica napus) and see how the control plants, those planted in soil with tungsten but without the bacteria, and the test plants, those with tungsten + bactera, differ in their morphological characters. Apart from that I also plan to assay the activity of the peroxidase enzyme, measure the free proline and total phenol in the test and control. This should give me a basic idea about the bioremediation capabilities of my bugs, or do you think it will Sir? Sir, the mine from where I sampled my soil had the scheelite ore, which is calcium tungstate. But I am using sodium tungstate...will that affect considerably? 

 

@El Crazy Xabi Sir, please elaborate on what is meant by a movable form of tungsten. I have actually never come across this terminology before. Sir, the culturing temperature has been around 35-37 degrees C. I do intend to use my bacteria as a biosorbent system. Sir, please let me know if figuring out the content of tungsten by atomic absorption spectroscopy of a broth culture inoculated with bacteria is an ok way to screen out the tungsten accumulating bugs or not. I intend to detect the levels of tungsten (through AAS) after 24 hours of inoculation and then finally at 74 hours of inoculation. The broths that show a decrease in W at 72 hours as compared to the original W conc would be selected for the bioremediation experiment. Sir, does it sound okay? And Sir unfortunately, I am unable to access the full papers of the references that you sent to me. 

 

I am figuring out a way to attach the picture of the green M9 Broth!


Edited by p.phadtare, 26 February 2014 - 09:37 AM.


#13 El Crazy Xabi

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Posted 26 February 2014 - 07:21 PM

Sodium tungstate is a soluble salt while scheelite is practically not soluble (ppm levels) unless you use acids. That affects to the mobility and bioavailability of the element. The total concentration of an element in a soil may be important but in remediation other fractions as extractables by different methods are also analysed. Any book on soil pollution and/or remediation or soil characterisation handbook should have all this information.

 

If you culture the bugs at 35-37° C it is OK if you are gonna use them as a biomass sorbent as they are not that likely to proliferate in soil, that temp is really hot for a soil, but also depends on the climate regime.... Releasing broth into the soil I don't think is a good idea. It is high in nutrients and may reach aquifers and cause eutrophication.

 

For the accumulation, do you use a control with W uninoculated? Measure that as W concentration reference. Sometimes you may get adsorption to the flask or precipitation, and evaporation is a common cause that also changes the concentration, not only the bacteria. But in this case it would be dependent on the amount of biomass (cell density) in your culture, not on their efficiency on absorption/uptake!! For that, you should expose a known amount of biomass (usually given as dry weight basis) under a given W concentration in a osmotically balanced and maybe buffered solution (it can be a mineral base solution used for mineral media preparation; e.g. M9 without C-source nor trace elements)... but avoiding any combination that might cause chemical precipitation, e.g. I cannot use phosphate containing solutions when I test U because it precipitates, which happens with most non-alkaline cations.

 

By the way... do you plan on remove the bugs from the soil after treatment? Usually biosorbents are use for treating running waters, aquifers or subsuperficial plumes where they are packaged and removed after treatment. Keep in mind that the behaviour of solutions in the lab may be relevant to aquifer decontamination but soils are completely different.

 

If you need a paper send me a pm



#14 p.phadtare

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Posted 28 February 2014 - 08:37 AM

Sir, I kept the culturing temperatures at around 37 degrees as it gets really hot at the place where the mines are situated. The temp touch 48 degress in peak summers. Sir, since scheelite is not soluble, then do you think I would have isolated any relevant bugs? Because I used sodium tungstate to screen them out. 

 

As for the addition of broth directly into the soil, could I dilute it and then release it? Also Sir, I intend to autoclave the soil before adding in the broth, do you think it will reduce the chances of eutrophication?

 

Sir for bioremediation, I am going to use an uninoculated W concentration as reference. Sir, before I proceed for the bioremediation experiment, I will inoculate a dried biomass of my bugs in the M9 broth, (without a C source) as you said, with a pre-decided conc of tungsten at which my bugs would remain viable and grow (will do MIC to determine the right W concentration). Then with the help of atomic absorption spectroscopy I will determine which inoculated broths show a decrease in W conc after 24 and 72 hours.  

 

Sir, here's a bit of a problem that I would like you to help me out with. How should I separate out my bug's biomass from the inoculated M9 broth cultures without bursting them? I wish to collect only the broth without the cells after 24 and 72 hours of inoculation to check the W conc in it. Should I simple centrifuge them at low rpms, like at around 2000 rpm? So that my cells do not burst and release whatever W they might have accumulated back into the broth that will be collected for AAS analysis? 

I will make sure I don't use a broth that may cause precipitation of W. 

 

No Sir, I do not plan on removing the bugs from the soil after treatment. This is actually only a preliminary analysis that I am doing. 

 

Sir could you please provide me with the brewery yeast as biosorbent paper? Also the following paper.

 

 Enterococcus faecalis strain LZ-11 isolated from Lanzhou reach of the Yellow River is able to resist and absorb Cadmium. by Wu G1Sun MLiu PZhang XYu ZZheng ZChen YLi X.

 

P.S. By the way Sir, I am really very grateful to you for all the help that you are providing me with. I would have hit a roadblock,  had it not been for this guidance. :)



#15 p.phadtare

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Posted 28 February 2014 - 08:51 AM

Arsenic accumulating bacteria isolated from soil for possible application in bioremediation.
 
Sir, if you could provide me with this paper too, then it would be great! :D





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